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Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by 1) adding long branched hydrophilic linkers between the substrate and the surface, 2) changing the orientation of peptides relative to the surface, and 3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates.
The human genome encodes approximately 500 kinases that regulate intracellular signal transduction, gene transcription, and cell development1. Changes in protein kinase activities, including changes in the selectivity and frequency of substrate phosphorylation, are implicated in human diseases ranging from autoimmunity to malignancies2-4. Given the association between changes in kinase activity and disease, many targeted pharmaceutical therapies are based on the modulation of kinase activities with small-molecule inhibitors5-9. Small-molecule inhibitors are initially screened for activity against kinases in large chemical libraries. Large libraries require efficient assay techniques for comparing inhibitor efficacies. A significant challenge in the development of high-throughput screening technologies is the measurement of altered kinase activities in their native cellular environment10, 11. In vitro assays using purified recombinant kinases that are truncated or epitope-tagged do not provide the molecular or cellular context to observe regulatory mechanisms affecting inhibitor efficacy. On the other hand, in vitro assays using cell lysates are both amenable to high-throughput formats and provide native kinases in an environment that approximates the complexity of intracellular conditions.
Substrate design is an important consideration in kinase assay development. In its most simplified form, a kinase assay monitors the phosphorylation of a protein or peptide substrate in the presence of a kinase and ATP. Kinase catalytic domains typically recognize a substrate tyrosine, serine, threonine, or histidine residue in the context of a short recognition motif composed of 3-20 amino acids12. These short motifs can be reproduced as independent peptides and used in place of intact protein substrates. Protein substrates can be difficult to produce in large amounts and peptides offer several advantages that simplify assay construction13-15. Compared to proteins, peptides are 1) relatively inexpensive to synthesize in large amounts, 2) easy to store, handle, and characterize, and 3) easily modified for immobilization or fluorescent detection. Peptide libraries are used to experimentally determine substrate sequences that provide the highest activity and specificity for a particular kinase in vitro16, 17. Although substrate specificity is generally more difficult to achieve with short linear sequences, peptides provide efficient tools for monitoring kinase acitivty18, 19. Peptide substrates are designed using experimentally derived consensus motifs20 and random peptide libraries21, 22 that are screened to identify substrate sequences with the highest rate of reactivity and degree of specificity. Peptide ligands for non-catalytic kinase domains23 have been presented in combination with peptide substrates to promote selective substrate phosphorylation. These developments have resulted in a recent increase in the use of highly multiplexed peptide arrays to investigate kinase activity in cell lysates24-27. Working with a small group of related peptides, this review focuses on strategies that optimize the design and presentation of peptide substrates to monitor kinase activity in cell lysates.
Whether derived from native recognition sequences or synthetically optimized, peptide substrates are used to screen for small-molecule inhibitors against kinases that are associated with disease (Table 1)17, 28-39. Abltide (EAIYAAPFAKKK)17 is one example of an optimized substrate that was selected by screening a degenerate peptide library against purified kinases in vitro. Abltide demonstrated a higher rate of reactivity with Abl kinase than with closely related kinases such as Src17 and is now routinely used to monitor Abl kinase activity in vitro. Abltide is preferentially phosphorylated by the Abelson subfamily, which includes Abl (ABL1) and Arg kinases (ABL2)40, as well as the oncogenic fusion protein Bcr-Abl41-44 that characterizes chronic myelogenous leukemia (CML). Accordingly, Abltide can be used to report on Bcr-Abl kinase activity in cell lysates, possibly providing an activity-based molecular diagnostic for CML. The specificity of Abltide as a reporter for Bcr-Abl activity can be confirmed by both the selective inhibition of Bcr-Abl and comparisons against cell extracts that demonstrate little Abl or Bcr-Abl kinase activity. Indeed, Abltide phosphorylation is quantitatively reduced with the addition of the Bcr-Abl inhibitor imatinib, the first FDA-approved tyrosine kinase inhibitor for the treatment of CML45 (sold as Gleevec by Novartis, East Hanover, NJ, USA). Similarly, cell lines such as the bone marrow-derived BaF3 line46, demonstrate that Abltide is minimally phosphorylated when Bcr-Abl is not expressed44. Assays that use Abltide as a reporter and imatinib as an inhibitor provide a model system for deriving general principles for the design of other peptide-based kinase assays.
The optimal modification of a peptide substrate depends in large part on the type of kinase assay in which it is used. Kinase assays can be broadly categorized into homogeneous (solution-phase)47, 48 and heterogeneous (solid-phase) technologies49-51. Homogeneous assays are typically small volume reactions using a purified enzyme and do not include post-reaction wash steps to reduce background52, 53. Heterogeneous kinase assays immobilize substrates on a two- or three-dimensional surface and detect phosphorylated products after multiple wash and isolation steps. Few homogeneous assays have been developed for use with cell lysates because signal-to-background ratios become prohibitively low due to non-specific interactions with intracellular proteins. However, unique applications of homogenous assays using cell lysates have presented peptides as direct reporters of kinase activity that do not require additional reagents, such as antibodies, to detect phosphorylation54. In two examples peptide substrates were modified with fluorophores that react with the phosphorylated amino acid55 or chelate co-factor metal ions present at the kinase active site during catalysis56. Heterogeneous assays, on the other hand, have the advantage of increasing the sensitivity for a specific reaction in cell lysates by isolating the product from other phosphorylated proteins in the cell.
Peptide substrates can be modified in a variety of ways for use in heterogeneous assays, although different immobilization strategies require specific considerations. Supporting surface geometries for peptide immobilization range from microarray chips and multi-well plates to micro- and nanoparticles. Peptides can be synthesized in situ on solid surfaces with photolithography or SPOT technology57, 58. Functional groups on peptide side chains have also been exploited for covalent immobilization on prepared surfaces. For example, primary amines have been immobilized on surfaces coated with epoxy, aldehyde, and N-hydroxysuccinimide functional groups. Similarly, amino-terminal cysteines have been used to react with glyoxylyl glass, forming a thiazolidine ring59, and with thioesters for native chemical ligation products60. Non-covalent attachment involves the use of biotin or poly-histidine tags61, while covalent methods have included click chemistry, such as Staudinger ligation62 or 1-3 dipolar cycloaddition63, and Diels-Alder reactions to photo-activated self-assembled monolayers on gold surfaces64 (also reviewed in65, 66). In contrast to proteins, which are difficult to immobilize in their active conformation67, 68, peptides are well suited to chemical modification and resistant to rigorous washes. Kinase assays using cell lysates require the addition of protease inhibitors to prevent the degradation of endogenous intracellular proteins and exogenous peptide substrates. Amino-terminal acetylation of peptides with acetic acid also provides improved stability in assays that use cell lysates69. Depending on the particular scheme that is chosen for peptide immobilization various modifications can be considered to improve peptide stability and accessibility.
The most common format for heterogeneous kinase assays is as an enzyme-linked immunosorbent assay (ELISA). Kinase assays with an ELISA format are easily constructed and performed without expensive equipment. In a typical ELISA, the protein or peptide substrate is immobilized on the surface of a multi-well plate either before or after reaction with a kinase. The phosphorylated substrate is probed with a phospho-specific primary antibody and an enzyme-conjugated secondary antibody. Upon addition of a suitable substrate for the conjugated enzyme, the chromogenic, luminescent, or fluorescent readout is quantified70. This method of labeling phosphorylated substrates can include the use of fluorescently labeled antibodies71 as well as the use of biotinylated reagents and their recognition with fluorescently labeled streptavidin. For example, fluorescence imaging using biotinylated Phos-tag and streptavidin has been used to detect the phosphorylation of peptide substrates immobilized to amino-modified glass via amino-terminal cysteines with glutaraldehyde crosslinkers72. Chemifluorescent detection, based on the oxidation of Amplex Red with hydrogen peroxide by horseradish peroxidase, has also been used to measure the relative phosphorylation of a peptide immobilized onto a hydrogel surface73. In this case, Abltide (CEAIYAAPFAKKK)17 was covalently attached by Michael addition of the amino-terminal cysteine to acrylamide-presenting hydrogel surfaces (Figure 1). C-Abl and Bcr-Abl kinase activities were monitored in cell lysates treated with a focused library of small-molecule inhibitors with pyridopyrimidine-like structures resembling imatinib73. Phosphorylated Abltide was detected with an anti-phosphotyrosine antibody, a horseradish peroxidase (HRP)-conjugated secondary antibody, and chemifluorescent imaging. The significant signal amplification that accompanies immuno-detection with enzyme-conjugates capable of producing fluorescent products yields an assay that is sensitive to small amounts of substrate phosphorylation but proportional to kinase activity.
Despite the general utility of the basic ELISA format, several features can be improved to increase the signal-to-background ratio in kinase assays using cell lysates. Surfaces often require passivation to decrease non-specific interactions with abundant proteins in cell lysates and peptide substrates require distance from the surface to improve kinase accessibility. This review will summarize the effects of increasing peptide substrate accessibility while decreasing non-specific interactions for improved reaction conditions on solid surfaces in kinase assays using cell lysates.
Surface passivation generally involves blocking the interaction surface with non-reactive molecules, such as BSA, casein, detergents and/or hydrophilic polymers, or capping un-conjugated functional groups such as reactive thiols with sodium acrylate. However, by using bioinert hydrophilic linkers to increase the distance between the peptide substrate and the surface, substrate accessibility can be improved while simultaneously decreasing non-specific interactions. For instance, a molecular layer of BSA74 or long polyethylene glycol linkers59 are commonly used to distance peptides from a surface. Branched linkers, which present chemical functional groups at multiple distal termini, improve the signal-to-background ratio by increasing substrate loading capacity. Comparisons between di-, tri-, and tetra-acrylate polyethylene glycol cross-linkers (Sartomer, Exton, PA, USA) demonstrated that the longest linker with three functional groups (SR415), yielded the strongest signal in a chemifluorescent ELISA using peptide substrates73. By increasing the density of peptide substrates on the hydrogel surface, the triacrylate linker increased the effective concentration of substrate in the hydrogel and improved readout sensitivity (Figure 2).
When immobilized, the relative rate of peptide phosphorylation is dependent on the accessibility of the substrate residues to kinases in solution. The core sequence of Abltide was optimized for maximal recognition by the catalytic sites of c-Abl and Bcr-Abl. However, when Abltide was immobilized in the kinase assay described above, the tyrosine was not optimally oriented for phosphorylation by approaching kinases (Figure 3A). To investigate the effects of distancing the substrate tyrosine from the site of immobilization, additional flexible and non-reactive amino acids, such as glycine and serine, were inserted near the site of immobilization (Figure 3B). Peptides C-4X-AT, C-6X-AT, C-8X-AT were synthesized with four, six, or eight amino acids between the amino-terminal site of immobilization and the tyrosine to be phosphorylated (Table 2). Although the maximum level of tyrosine phosphorylation increased relative to the original sequence, the signal did not increase proportionately with additional amino acid spacers (Figure 4). An eight amino acid spacer and a four amino acid spacer resulted in the same relative degree of tyrosine phosphorylation. To investigate the influence of Abltide orientation in the context of a passivated hydrogel, the site of immobilization was reversed from the amino-terminus to the carboxyl-terminus. Peptides were synthesized with cysteines at the carboxyl-terminus (AT-C) and compared to peptides with four additional amino acids at the distal end of the peptide (4X-AT-C) (Figures 3C and 3D). In both cases, there was increased phosphorylation relative to Abltide immobilized at the amino-terminus (Figure 4). From these examples, two general principles can be derived to improve the degree of substrate phosphorylation in heterogeneous assays: 1) increase the relative distance between the substrate and the site of immobilization, and 2) present the substrate tyrosine in an orientation that allows kinase accessibility.
C-Abl and Bcr-Abl are structurally related to the Src family of kinases and use SH2 and SH3 domains to regulate inter- and intra-molecular interactions75. While the catalytic kinase domain is responsible for the phosphorylation of substrates, SH2 and SH3 domains guide the kinase to its substrates and regulate its active state76. By providing only a short amino acid sequence that is recognized exclusively by the kinase catalytic site, peptide substrates often lack the selectivity shown to protein substrates. Adding a peptide ligand for the SH2 or SH3 domains can significantly improve substrate recognition thereby increasing the relative rate of substrate phosphorylation77, 78. In heterogeneous kinase assays, where peptide substrates are immobilized in relatively inert environments, the inclusion of peptide ligands for SH2 and SH3 domains effectively recruits cellular kinases to the surface. In the ELISA format previously described using cell lysates to phosphorylate Abltide on a hydrogel surface, the co-immobilization of a high-affinity peptide ligand for the SH3 domain of c-Abl (p40; CGGAPTYSPPPPPLL)79 significantly increased Abltide phosphorylation. Although Abltide peptide substrate and the p40 SH3 ligand were not synthesized in tandem but were immobilized in parallel on the hydrogel surface (in trans), the presence of the SH3 ligand appeared to increase the effective concentration of kinase at the hydrogel surface73. Similarly, by expressing Abltide in tandem with p40 (in cis) as a carboxyl-terminal tail to glutathione-S-transferase and immobilizing on glutathione agarose beads44, Abltide phosphorylation was increased. In theory, the combination of kinase ligands and peptides in trans or in cis can be applied to any kinase with multiple recognition domains76. Using current synthesis techniques such as native chemical ligation80, 81, it is possible to assemble libraries of optimized substrates82, or “super-substrates,” constructed from the modular rearrangement of specialized peptides for recognition by particular domains.
Solution-phase heterogeneous kinase assays aim to improve the reactivity of solid-supported peptide substrates by providing greater access to reactants in solution. Despite improvements in the presentation of peptides on a solid surface, the reaction rate between immobilized peptides and kinases in solution can be slow due to the limits of diffusion at the interface83, 84. In contrast to many two-dimensional plate-based assays, three-dimensional bead-based assays can use mechanical mixing to increase the frequency of molecular interactions at the interface between solid and solution phases. Uniformly distributed in a solution of reactants, beads bearing peptide substrates display faster apparent reaction rates with kinases in solution. Furthermore, a dense suspension of sub-µm beads can have a larger total surface area per volume than a flat two-dimensional surface overlaid with solution (>1 μl to wet 1 cm2) and each bead can be loaded to higher capacity with peptide substrates due to bead surface curvature. Because the density of immobilized substrate in a given reaction volume limits the maximum signal obtainable in a heterogeneous assay, higher substrate loading with better accessibility results in a higher signal-to-background ratio. Using immobilization strategies similar to those used for plate-based assays, but with shorter processing times and easier handling procedures, beads offer the advantages of high loading capacity, increased apparent reaction rates, and improved substrate accessibility to kinases in solution.
Multiple peptide substrates can be presented in a shared reaction volume using beads that can be individually distinguished by internal fluorescence (e.g. Luminex liquid bead arrays, Luminex, Austen, TX, USA). Peptide-conjugated beads can be detected by a flow cytometer after labeling with fluorescent phospho-specific antibodies. For example, a mixture of purified kinases was used to phosphorylate biotinylated peptide substrates immobilized on streptavidin-conjugated fluorescent beads85. This strategy of using uniquely identifiable beads to detect multiple substrates was broadened to investigate the competitive phosphorylation of substrates by a kinase in cell lysates. Using the same chemistry that was developed for two-dimensional hydrogel surfaces, Abltide was immobilized in two orientations (C-AT and AT-C) on carboxyl-coated fluorescent beads via Michael addition of a cysteine to an amino-acrylamide linker. The peptide ligand for the Abl kinase SH3 domain, p40 (AL), was included in cis at the amino or carboxyl-terminus of Abltide (C-ALAT and C-ATAL). To measure the degree of substrate phosphorylation, four peptide standards were included in each reaction volume after the kinase reaction and before labeling (Figure 5). Antibody-based fluorescence intensity was translated to the percentage of substrate phosphorylation by non-linear regression from well-specific internal standard curves (Figure 6A). Presented in a single reaction volume, substrates that included the p40 c-Abl SH3 ligand sequence were competitively phosphorylated by Bcr-Abl in cell lysates over peptides that presented Abltide alone (Figure 6B). Assay conditions can be optimized to improve sensitivity and the limits of detection by using a combination of peptide ligands and internal peptide standards. The advantage of methods such as fluorescently encoded bead arrays that allow the multiplexed presentation and detection of peptide substrates is that substrate efficiency can be quantitatively compared in a single reaction.
Whether applied to peptides immobilized on hydrogel surfaces or Luminex beads, ELISA-based methods depend on the availability of antibodies or similar reagents for the selective recognition of distinct peptide sequences or phosphorylated residues. On the other hand, label-free methods provide readouts that are independent from additional reagents used for detection. For example, mass spectrometry (MS) uses a readout based on the abundance of ions to detect distinct differences in peptide mass before and after substrate phosphorylation. This can be applied to the detection of peptides in solution using liquid chromatography (LC)-MS27 or to immobilized peptides using matrix-assisted laser desorption ionization (MALDI) MS86. In one example using peptide substrates immobilized on gold surfaces, self-assembled monolayers for MALDI (SAMDI)64 provided a semi-quantitative and unambiguous measure of substrate phosphorylation. As an alternative to MALDI detection with higher sensitivity for surface-based analyses, time-of-flight secondary ion MS (TOF-SIMS) was used to monitor peptide phosphorylation by purified kinases in vitro87. Conjugating peptides to gold nanoparticles clustered on amine-coated glass increased loading capacity and substrate accessibility on the two-dimensional glass surface88. This demonstrates that despite the absence of additional labeling reagents, heterogeneous kinase assays using label-free methods benefit from optimized peptide immobilization and surface passivation.
Most MALDI-based methods do not include a separate step for the release of immobilized peptides, instead relying on energy imparted by the laser to desorb peptides from the solid surface. However, to control the release of immobilized peptide substrates prior to analysis with label-free MS an ultraviolet (UV)-cleavable substrate was included89. Using a modular approach to substrate synthesis based on native chemical ligation, a photocleavable linker was incorporated at the amino-terminus of Abltide followed by p40, the peptide ligand for the Abl SH3 domain. This UV-cleavable substrate was co-polymerized with bis-acrylamide to construct a hydrogel matrix of Abltide-functionalized polymers. With three-dimensional substrate loading capacity, hydrogel matrices swell upon the addition of soluble reactants, thereby providing a sturdy and inert environment for biochemical reactions. After substrate phosphorylation the linker was cleaved by UV light and Abltide was released for MALDI-TOF detection. Compared to peptide recognition in an ELISA format, the benefit of using mass spectrometry is the unambiguous detection of substrate mass. This allows multiple peptides with distinct masses to be analyzed in a single sample. Therefore by including an internal “heavy” peptide control, the relative intensities of phosphorylated and un-phosphorylated Abltide were quantitatively determined from a single sample using label-free detection (Figure 7A, unpublished data, Xiangfu Shi).
With label-free detection, multiple peptide substrates can be used to monitor different kinase activities from purified mixtures or cell lysates. For example, MALDI-TOF can be used to detect the selective phosphorylation of two peptides immobilized on a hydrogel surface by two kinases in a mixture. Un-phosphorylated and phosphorylated forms of each peptide are detected, allowing a straightforward qualitative analysis of reaction components (Figure 7B, Xiangfu Shi). To more accurately compare MALDI detection to chemifluorescent ELISA, a dual-detection kinase assay was developed using biotinylated Abltide immobilized on streptavidin-conjugated magnetic beads (unpublished data). Used to screen for small-molecule inhibitors, the dual-detection kinase assay eliminates false positives and negatives by validating results using two separate detection and readout methods. More complex signaling networks may be investigated using label-free methods to detect multiple peptide substrates on a shared surface in kinase assays using cell lysates. These applications may lay the foundations for using intracellular signaling profiles to personalize targeted therapies with kinase inhibitors in clinical settings90.
Kinase assays that use cell lysates instead of an isolated kinase of interest more accurately approximate in vivo responses. To investigate the efficacy of a small-molecule inhibitor, it is necessary to account for environmental cues that regulate kinase activity. Kinase assays using cell lysates are complicated by the difficulty of monitoring a specific enzyme activity within a complex biological sample. Heterogeneous formats facilitate the separation of products from extraneous cell components and offer the advantage of reducing non-specific background with wash steps. Using peptide substrates in heterogeneous kinase assays simplifies substrate immobilization strategies and assay analysis. Changes in chemical modifications and physical orientations can be used to optimize substrate presentation for improved sensitivity and specificity in kinase assays using cell lysates.
This review summarizes several chemical approaches to peptide immobilization and emphasizes our efforts to optimize assay sensitivity in a variety of formats, including chemifluorescent ELISAs on hydrogel surfaces, antibody recognition on fluorescent beads, and with label-free methods such as MS. Chemical modifications that affect the orientation and distance of a peptide from a passivated surface result in improved substrate accessibility and recognition. This allows kinases to phosphorylate a higher concentration of substrates and produces better signal-to-background ratios. In several examples varying the orientation of peptide substrates on a surface and using long, branched hydrophilic linkers increased the degree of substrate phosphorylation and decreased non-specific interactions at the surface. In addition, the inclusion of peptide ligands for non-catalytic kinase domains such as the p40 ligand for the Abl kinase SH3 domain improved substrate phosphorylation by directing kinases from solution to the surface. Indeed, on both hydrogels and beads, modular rearrangements of the Abltide and p40 sequences increased Abltide phosphorylation by Bcr-Abl kinase in cell lysates. To reveal kinase substrate preferences, peptides were multiplexed using fluorescent Luminex beads and competitively phosphorylated by cell lysates. The inclusion of internal peptide standards for quantitative analysis strengthened results obtained from a single multiplexed experiment. Separate beads can be used to immobilize unique peptides reporters for the simultaneous measurement of multiple phosphorylation events.
Label-free detection with MS provided a more flexible platform for the development of multiplexed assays. Although previously applied to purified kinase activities in vitro, promising developments include the use of MALDI-TOF MS to monitor kinase activity and inhibition in cell lysates with peptides immobilized on magnetic beads or glass slides89. With the advantage of unambiguous results, reversible linkers and internal peptide controls can be included to quantitatively analyze kinase activity by MALDI-TOF. Toward this end, a dual-detection kinase assay was developed using both MALDI-TOF and chemifluorescent ELISA to compare readouts and eliminate false positives and negatives. Dual-detection methods may speed up the screening process thereby lowering the cost of high-throughput screens.
Although this review highlights the detection of Bcr-Abl activity in a model of CML using the peptide substrate Abltide, the strategies discussed in this review can be generalized for most peptide substrates in heterogeneous kinase assays using cell lysates. Advances in peptide synthesis technology and robotics for rapid handling will undoubtedly influence the popularity of peptide substrates in heterogeneous kinase assays. However, more compelling developments may be brought about by improved immobilization strategies that allow the presentation of optimized peptide substrates in high density for small volume reactions with cell lysates.
We congratulate Professor Stephen B. H. Kent on receiving the Merrifield Award at the 21st American Peptide Society Symposium. We acknowledge our collaborators Sean Palecek, Alex Schilling, Wendy Stock, Ezra Abrams, Stephen Kent, David Angulo, and Darren Veach for their contributions to these studies. We thank our colleagues Xiangfu Shi, Evan Nair-Gill, Joon Huh, Aaron Engel-Hall, Jennifer Campbell, Sandra Korn, Shariska Petersen, Britton Walker, Mariah Siddiqui, George Steinhardt, Michael Mand, Won Jun Rhee, Stacey Kigar, Eric Yan, and Vivian Tien for their efforts to develop the approaches reported here. Financial support was provided by the NIH R33 CA103235 (S.J.K), R01 HG003864 (S.J.K.), R21 CA126764 (S.J.K.), R01 GM074691 (to S. Palecek), K99 CA127161A (L.L.P.), NSF University of Chicago MRSEC (S.J.K.), and the American Heart Association Midwest Affiliate predoctoral fellowship (J.E.S). S.J.K was a Leukemia & Lymphoma Society Scholar.
The National Institutes of Health