Timed pregnant female and 8- to 10-week-old male C57BL/6 mice were obtained from Charles River Laboratories and maintained under sterile barrier conditions. All procedures were reviewed and approved by the University of California IACUC.
Human Cx43 and human EB1 cDNAs were obtained from Open Biosystems and cloned into pDONR/Zeo (Invitrogen) using Gateway BP cloning to generate entry (ENTR) clones. Cx43 and EB1 genes were subsequently inserted into pDest-EGFP-N1, pDest-TRE-Tight (converted vectors originally from Clontech), pcDNA3.2-V5-Dest, pcDNA3.1/n-V5, and pLenti6.3-V5-Dest (Invitrogen) by Gateway LR cloning. Human Cx43 (with a stop codon) was also inserted by gateway cloning into pLenti6.3/V5-Dest. The EB1-K89E point mutation was generated using the Quickchange II site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions and pENTR/Zeo-EB1 as a template (forward primer: AGAATGGGTGTTGACGAAATAATTCCTGTGGAC; reverse primer: GTCCACAGGAATTATTTCGTCAACACCCATTCT). EB1 was inserted into pLVX-AcGFP-N1 (Clontech) by traditional restriction cloning.
HeLa cells (ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 100 μg/ml Normocin (Amaxa). Primary mouse neonatal cardiomyocytes were isolated from p3 C57BL/6 mice and maintained in F12/DMEM 50/50 (Invitrogen) supplemented with 2% FBS, insulin–transferrin–sodium selenite media supplement, 10 μM BrdU, 20 μM cytosine β-d-arabinofuranoside (Sigma-Aldrich), and 100 μg/ml primocin (Amaxa Biosystems). Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. Unless otherwise stated in the figure legends, cells were seeded at a density of 7 × 104 cells/cm2 and allowed to adhere overnight. Cells were treated with 200 μM H2O2 (Sigma-Aldrich) in fully supplemented medium prior to fixation.
Generation of pLenti6.3-Cx43 clonal HeLa cell line.
Lentivirus was produced from pLenti6.3-Cx43 according to the manufacturer’s (Invitrogen) instructions and used to infect HeLa cells previously plated in 6-well dishes in the presence of 4 μg/ml hexadimethrine bromide (Sigma-Aldrich). Medium was changed the following morning, and cells were split into 100-mm dishes at a dilution of 1:40 in the presence of blasticidin (10 μg/ml). Medium was changed every 2 days and healthy colonies were picked using trypsin cloning cylinders (Scienceware), expanded, and screened for expression by Western blotting and immunofluorescence.
EB1 and α-tubulin immunofluorescence.
For EB1 and α-tubulin staining, cells were fixed for 5 minutes at –20°C in 100% methanol. Three PBS washes were performed, and cells were permeabilized for 4 minutes using 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Cells were then washed and blocked for 1 hour at room temperature in 5% normal goat serum (NGS; Invitrogen) before addition of primary antibodies. Mouse monoclonal antibodies to EB1 (1:500) were obtained from BD Biosciences, and rat monoclonal antibodies to α-tubulin (1:500) were from Abcam. Cells were incubated with primary antibodies diluted in 5% NGS in PBS for 1 hour at room temperature. Following several PBS washes, cells were incubated for an additional hour with goat secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen), or CY3 (Jackson Laboratories) and TO-PRO-3 nuclear counterstain (Invitrogen). Cells were washed with PBS, washed briefly with dH2O, and coverslips were mounted using ProLong gold antifade reagent (Invitrogen). Slides were allowed to dry overnight and imaged using a Nikon Ti microscope with a ×60/1.49 Apo TIRF objective, Yokogowa CSU-X1 spinning disk confocal unit with 486-, 561-, and 647-nm DPSS laser source, and Coolsnap HQ2 camera controlled by NIS Elements software (Nikon). Additional image processing and analysis was performed using ImageJ (NIH). Equal thresholds were set for individual images, and EB1 comet length was measured blind using the ImageJ measure function. Only those comets approaching the cell periphery and entirely within the focal plane were measured. At least 15 comets/cell were measured for 5 cells within each condition.
Surface protein biotinylation.
pLenti6.3-Cx43 HeLa cells were seeded in 100-mm dishes and allowed to adhere overnight. Cells were incubated for 16 hours in the presence or absence of 200 μM H2O2. Dynasore (80 μM; Sigma-Aldrich) was added 1 hour prior to H2O2, and DMSO was added as vehicle to control cells. Freshly isolated primary adult mouse ventricular cardiomyocytes were seeded in fully supplemented Cardiac Myocyte Medium (CMM; ScienCell) onto 600-mm dishes that had been coated overnight with 2 mg/ml laminin (Invitrogen). Cells were allowed to adhere for 1 hour before exposure to 10 μM H2O2 or not diluted in fresh CMM in the presence or absence of 40 μM Dynasore for 4 hours. Surface proteins were biotinylated following 2 washes with ice-cold PBS by incubating cells for 20 minutes at 4°C with EZ-link Sulfo-NHS-SS-Biotin (Pierce Biotechnology) at a concentration of 1 mg/ml in PBS. Cells were washed in ice-cold PBS and incubated twice for 5 minutes each time in PBS containing 100 mM glycine to remove unbound biotin. After a further 3 washes in ice-cold PBS, cells were lysed using 200-μl RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 2 mM NaF, 200 μM Na3VO4) supplemented with Complete Mini Protease Inhibitor Cocktail (Roche). Cells were scraped into RIPA buffer, and lysates were sonicated using a Microson ultrasonic cell disruptor (Misonex) before centrifugation at 10,000 g for 20 minutes at 4°C. Protein concentration in the clarified lysates was determined using the BioRad DC Protein Assay and normalized. A total of 1 mg of protein from each condition was incubated in RIPA buffer with 25 μl of Ultralink Immobilized NeutrAvidin slurry (Pierce Biotechnology) overnight at 4°C with rotation. Tubes were centrifuged for 1 minute at 300 g and allowed to settle for 2 minutes before the supernatant was removed. RIPA buffer (1 ml) was added to each tube, and tubes were inverted to mix and spun down again at 300 g. This process was repeated 4 times before the addition of 20 μl 2X NuPAGE sample buffer (Invitrogen) supplemented with 100 mM DTT (Sigma-Aldrich). Samples were heated for 10 minutes at 70°C prior to subjection to SDS-PAGE electrophoresis and Western blotting (see below).
After heating to 70°C for 10 minutes, samples were cooled to room temperature and subjected to SDS-PAGE electrophoresis using NuPAGE Bis-Tris gels and MES (Invitrogen) buffer according to the manufacturer’s instructions. Gels were transferred to FluoroTrans PVDF membranes (Pall), which were subsequently fixed by soaking in methanol and air drying before rewetting with methanol and blocking for 1 hour at room temperature in 5% nonfat milk (Carnation) in TNT buffer (0.1% Tween 20, 150 mM NaCl, 50 mM Tris pH 8.0). Membranes were then incubated overnight with primary antibodies diluted in 5% milk in TNT. Primary antibodies used were rabbit anti-Cx43 (1:4,000; Sigma-Aldrich), rabbit anti–N-cadherin (1:4,000; Abcam), rabbit anti–β-catenin (1:2,000; Abcam), rabbit anti-EB1 (1:4,000; Sigma-Aldrich), rat anti–α-tubulin (1:1,000; Abcam), rabbit anti-V5 (1:3,000; Sigma-Aldrich), mouse anti–β-actin (1:3,000; Sigma-Aldrich), and mouse anti-GAPDH (1:3,000; Abcam). Membranes were washed 5 times before incubation for 1 hour at room temperature with secondary antibodies diluted in 5% milk TNT. Goat secondary antibodies conjugated to Alexa Fluor 555 and 633 were used at a dilution of 1:1,000. Following incubation with secondary antibody, membranes were washed 5 times, soaked in methanol, and allowed to air dry. Membranes were imaged using the Versadoc MP 4000 fluorescent western detection system (BioRad).
Imaging was carried out using a Nikon TE-2000E inverted microscope with a ×100/1.49 Apo TIRF objective and through-the-objective TIRF illumination using a 488-nm argon laser for EGFP visualization and a 514-nm laser for EYFP. The photometrics Cascade II 512 camera was used for acquisition of all TIRFm studies. Cells were maintained in HBSS (Invitrogen) plus 10% FBS at 37°C for all live cell imaging studies. All TIRF image processing was performed with ImageJ software (NIH).
Assessing the effect of oxidative stress on the interaction of EB1-EGFP with the plasma membrane.
Glass-bottomed 35-mm dishes (Mattek) were coated with N-cadherin as previously described (24
). HeLa cells were plated in complete DMEM and allowed to adhere overnight. Cells were transfected with pDest-EB1-EGFP-N1 using 2 μg DNA/dish and Fugene 6. For primary neonatal mouse ventricular cardiomyocytes, dishes were coated with 5 μg/ml fibronectin and 0.02% gelatin for 4 hours prior to N-cadherin attachment. Lentivirus was produced using pLVX-EB1-AcGFP according to the manufacturer’s instructions (Clontech and Invitrogen), and cardiomyocytes were infected overnight in the presence of 4 μg/ml hexadimethrine bromide. Lentivirally transduced cardiomyocytes were imaged 72 hours after infection. Medium was changed to HBSS with 10% FBS (2% for neonatal cardiomyocytes) and epifluorescence used to select cells expressing similar amounts of EB1-EGFP 24 hours after transfection. TIRFm time lapse sequences of 2 minutes were acquired at an exposure of 200 ms per image and rate of 30 frames/minute. Acquisitions were performed every 15 minutes for 45 minutes under normal conditions, H2
was added to the HBSS to a final concentration of 200 μM, and 3 more acquisitions were performed at 15-minute intervals. At this point, dishes were gently washed 4 times with warm HBSS containing 10% FBS, and a final 3 acquisitions were taken to assess recovery over a 45-minute period. Comet density was determined by first defining a region of interest via a freehand tool on ImageJ, then background subtraction was performed with a 10-pixel rolling ball radius and thresholds were assigned to images. Comets were defined as 2 or more contiguous pixels identified by the particle analyzer function.
Live-cell spinning disc confocal microscopy of α-tubulin–EGFP.
HeLa cells were plated and transfected with pEGFP-N1–α-tubulin as described above. Twenty-four hours after transfection, cells were exposed to normal conditions or 200 μM H2
for 2 hours, after which dishes were imaged using a Nikon Ti
inverted microscope, Yokogowa CSU-X1 spinning disk confocal unit with 486-nm DPSS laser source, and a Cascade II 512 camera (Photometrics). Images were acquired every 5 seconds (400-ms exposure) for a time span of 5 minutes. Only microtubules that were observed growing and/or shrinking near the cell periphery for several minutes were tracked. The position of each microtubule end was tracked over time using ImageJ (NIH). Images were inverted, background subtracted, and contrast enhanced. The ImageJ plug-in MTrackJ (http://www.imagescience.org/meijering/software/mtrackj/) was used to manually track individual microtubule plus ends. Microtubule dynamics and events were classified as previously reported (64
) with an optical resolution limit of 0.16 μm and analyzed in Excel (Microsoft) using a custom-written macro. The number of microtubules directly approaching the cell cortex (within 3 μm of cell edge) was determined for each 5-minute acquisition. Data were compiled from 3 separate experiments to a total of 9 cells per condition.
Generation of tetracycline-inducible Cx43-EYFP HeLa cell clones.
HeLa Tet-ON cells were purchased from Clontech and maintained in fully supplemented DMEM containing 500 μg/ml Geneticin (Invitrogen). Cells were plated into 100-mm dishes at a density of 106 cells/dish and allowed to adhere overnight. The next day, the cells were transfected with pDest-TRE-Cx43-EYFP and a linear Hygromycin selection marker (Clontech) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were trypsinized and plated into fresh dishes at a dilution of 1:40 in fully supplemented medium containing 500 μg/ml Geneticin and 200 μg/ml Hygromycin. Dishes were monitored and when large healthy colonies had developed, clones were picked using trypsin cloning cylinders, expanded, and screened for tightly regulated expression.
Real-time detection of Cx43-EYFP delivery to the plasma membrane using TIRF microscopy.
Glass-bottomed dishes (35 mm) were coated with N-cadherin as previously described (24
) before clonal HeLa Tet-ON Cx43-EYFP cells were seeded at a concentration of 105
cells/dish and allowed to adhere overnight. Cells were washed once with PBS and induced with 2 μg/ml doxycycline (Sigma-Aldrich) in HBSS (Invitrogen) without phenol red supplemented with 10% FBS. Dishes were moved to the heated chamber of a Nikon Eclipse TE2000-E microscope and left undisturbed for 120 minutes. At this point, fresh supplemented HBSS containing doxycycline with or without H2
at a final concentration of 200 μM was added. After 30 minutes (t
= 150 minutes after induction), it was possible to see Cx43-EYFP in the ER and Golgi by epifluorescent detection. Healthy cells were selected, and TIRF microcopy was used to acquire 90-minute time-lapse sequences, with images acquired 2 minutes apart for 200 milliseconds per image. For quantification, ImageJ software was used to determine fluorescence intensity within a region of interest defined by the cell periphery.
Detection of Cx43-EYFP delivery to the plasma membrane by biotinylation.
HeLa Tet-ON Cx43-EYFP cells were seeded in 100-mm dishes at a concentration of 106 cells/dish and allowed to adhere overnight. Fresh medium containing 2 μg/ml doxycycline was added to the cells 120 minutes prior to incubation with H2O2 for 120 minutes at 37°C.
Zebrafish husbandry and oxidative stress induction.
Zebrafish were raised under standard laboratory conditions at 28°C. The following transgenic lines were used: Tg(cmlc2:cx48.5-EGFP)s882
). The cmlc2:Cx48.5-EGFP
construct was created by cloning a 900-bp fragment of the cmlc2
promoter upstream of the full-length cDNA zebrafish Cx48.5
fused to EGFP. Linearized DNA (200 pg) was injected into 1 cell–stage embryos, and we selected individual transgenic carrier adults by screening for fluorescent progeny. Three Tg(cmlc2:Cx48.5-EGFP)s882
founders were recovered with nearly identical expression patterns and levels. In order to induce oxidative stress on zebrafish embryos, H2
was serially titrated from 1 nM to 200 μM in egg water medium. Embryos were treated at 24 hpf and analyzed at 48–60 hpf.
Zebrafish immunohistochemistry and confocal microscopy.
Immunohistochemistry and confocal microscopy were performed as previously described (48
). Affinity-purified zebrafish cadherin 2 antibody (66
) was used at a dilution of 1:80.
Zebrafish optical mapping by widefield epifluorescence.
Using the Tg(cmlc2:gCaMP)s878
line, optical mapping was performed on individual embryos at 48–60 hpf as previously described (48
). Briefly, the upstroke of the calcium transient is registered as the activation point of the cell, which is appropriate if one assumes the same phase delay between voltage activation and calcium transient through the ventricle. Isochronal maps were drawn, and the activation time between the isochrones was recorded for a measurement of conduction velocity.
Langendorff-perfused mouse heart preparation.
Male C57BL/6 mice were anesthetized with isoflurane and injected with heparin (50 IU i.p.). After cervical dislocation, hearts were removed quickly by a midsternal incision and placed into ice-cold modified Krebs-Henseleit (K-H) solution. Under a dissecting microscope, the aortic opening was immediately cannulated and tied on a 23-gauge stainless steel blunt needle. The heart was attached to a Langendorff apparatus (ADInstruments) and perfused through the aorta at a constant rate of 4 ml/min with a modified pH 7.4 K-H buffer of the following composition (in mM): NaCl 118, KCl 4.7, CaCl2.H2O 2.5, MgCl2.7H2O 1.2, NaHCO3 24, KH2PO4 1.2, glucose 11, EDTA 0.5. The K-H solution was prefiltered by a microfilter (0.2-μm diameter; Nalgene) and constantly gassed with 95% O2/5% CO2. Perfusion medium was passed through water-jacketed tubing and cylinders, and the temperature was maintained at 37°C with a temperature-controlled circulating water bath. The hearts were allowed to equilibrate for 10 minutes to achieve a steady state before they were subjected to 30 minutes of global ischemia, followed by 60 minutes of reperfusion. Control hearts were perfused continuously throughout the protocol. Pacing was used (typically at 150% of threshold: 500 mA, Stimulus Isolator; ADInstruments) during the whole experimental protocol except for periods of ischemia. During no-flow ischemia, the heart was immersed in warm K-H buffer in order to maintain warmth and moisture. Immediately after Langendorff procedure, hearts were placed in cryovials and snap-frozen in liquid N2 for biochemical studies. For cryosectioning, hearts were embedded in OCT (Sakura Finotek) and snap-frozen by immersing in liquid N2-chilled isopentane to snap-freeze before storage at –80°C.
Co-immunoprecipitation from whole mouse heart tissue.
Frozen hearts were weighed and added to lysis buffer (50 mM Hepes pH 7.4, 150 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM NaF, 100 μM Na3VO4, 0.5% Triton X-100) to a final volume of 200 mg/ml. Tissue was homogenized for 10 seconds and incubated at 4°C with rotation for 1 hour before centrifugation at 10,000 g for 20 minutes to remove insoluble debris. Following protein normalization, lysates were precleared using Dynabeads protein G (Invitrogen) for 30 minutes at 4°C with rotation. Beads were discarded, and 1 mg of precleared lysate was used per reaction. Lysates were incubated for 60 minutes at 4°C with rotation with 2 μg of primary antibody or mouse anti-GST (Santa Cruz Biotechnology Inc.) as a negative control. Dynabeads protein G (15 μl) was added to each reaction, and tubes were rotated for a further 45 minutes at 4°C. Beads were washed 4 times for 5 minutes each time with 1 ml lysis buffer on ice using a Dynamag-2 magnet (Invitrogen). Proteins were eluted from beads in 10 μl 2X NuPAGE sample buffer and subjected to SDS-PAGE electrophoresis and Western blotting as described above.
Lipid hydroperoxide assay.
As a measure of oxidative stress in whole mouse hearts, the lipid hydroperoxide assay kit from Cayman Chemical Company was used. Immediately after Langendorff perfusion experiments, 20 mg of heart tissue was homogenized and chloroform lipid extraction performed as per the manufacturer’s instructions. HeLa cells were scraped into ice-cold water and sonicated prior to lipid extraction. The assay was performed in a glass 96-well dish and absorbance read at 500 nm using a Spectramax Plus plate reader (Molecular Devices).
Human tissue collection.
With the approval of the UCSF Committee for Human Research, we obtained tissue from hearts removed at the time of transplant at UCSF, or from organ donors whose hearts were not transplanted for technical reasons. Fully informed consent was obtained from all UCSF transplant recipients prior to surgery. The California Transplant Donor Network (CTDN) provided the unused donor hearts and obtained informed consent for their use from the next of kin.
Cold cardioplegia was perfused antegrade prior to cardiectomy, and the explanted heart was placed immediately in ice-cold physiologic solution. Full-thickness samples from the base of the left ventricle were cleaned rapidly of all epicardial fat and embedded in OCT medium. The embedded tissue was submerged in liquid N2-chilled isopentane to snap-freeze before storage at –80°C.
The donor hearts (non-failing) came from four males ages 62–67 without significant past medical history, who sustained irreversible neurological damage secondary to non-cardiac trauma.
The diseased hearts (failing) came from 3 males ages 19–51 and 1 female age 57, all with end-stage ischemic cardiomyopathy undergoing cardiac transplant at the time of tissue acquisition. Echocardiographic data from one of each patient group are presented in Supplemental Figure 5.
Cryosectioning and immunofluorescence of human and mouse heart tissue.
Cryosections (6 μm) were prepared and positioned on poly-L-lysine–coated slides prior to acetone fixation and air drying. Sections were outlined using a super PAP pen (Invitrogen) and rehydrated for 10 minutes in PBS. Following 1 hour of blocking at room temperature with 5% NGS in PBS, sections were incubated at 4°C for 16 hours with primary antibodies diluted in 5% NGS. Primary antibodies used were: Cx43 (rabbit, 1:3,000; Sigma-Aldrich), EB1 (rabbit, 1:3,000; Sigma-Aldrich), and N-cadherin (mouse, 1:500; BD Biosciences). Following several PBS washes, cells were incubated for an additional hour at room temperature with goat secondary antibodies conjugated to Alexa Fluor 488 or 555. For detection of N-cadherin in mouse tissue, the same monoclonal mouse anti–N-cadherin antibody was biotinylated using EZ-link sulfo-NHS-SS-biotin and Slide-A-Lyzer dialysis cassettes (Pierce Biotechnology) according to the manufacturer’s instructions. Secondary detection was achieved using Streptavidin Alexa Fluor 488 conjugate (1:200; Invitrogen) diluted in high-salt buffer (0.5 M NaCl, 10 mM Hepes). Slides were washed with PBS, briefly washed with dH2O, and coverslips were mounted using ProLong gold antifade reagent containing DAPI. Slides were dried overnight and imaged using a Nikon Ti microscope with a ×60/1.49 Apo TIRF objective, Yokogowa CSU-X1 spinning disk confocal unit with 486-, 561-, and 647-nm DPSS laser source, and Coolsnap HQ2 camera controlled by NIS Elements software.
Quantification of Cx43 and EB1 at adherens junctions in cryosections by immunofluorescence.
N-cadherin overall immunofluorescent signal intensity was found to be associated with overall sample quality, consistent with previous reports (57
). For this reason, acquired images were normalized relative to N-cadherin signal before quantif–ication. To isolate intercalated disc regions, ImageJ software was used to subtract background from N-cadherin images, to which equal thresholds were applied, generating binary masks. Within this mask image, intercalated disc regions had a value of 1 and all remaining pixels had a value of 0. Masks were image-multiplied by corresponding Cx43 or EB1 images, to exclude all signal except that at the intercalated disc, and fluorescence intensity was subsequently measured. To determine the percent enrichment of EB1 or Cx43 at intercalated discs relative to overall signal, intercalated disc signal intensities were divided by the overall average signal of EB1 or Cx43 in the image. For each sample, 6 randomly selected images containing, on average, 31 intercalated discs were analyzed.
Extraction of human heart soluble (non-junctional) and insoluble (junctional) proteins.
Snap-frozen tissue samples were weighed and added to 1% Triton X-100 buffer (50 mM Tris pH 7.4, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 250 mM NaCl, 1 mM NaF, 0.1 mM Na3VO4, Complete Mini protease inhibitors) at a final concentration of 100 mg tissue/ml. Samples were homogenized and nutated for 1 hour at 4°C. At this point, 300 μl of lysate was removed and added to an equal volume of 1% Triton X-100 buffer containing 8 M urea and 2 M thiourea (Sigma-Aldrich) in order to solubilize junctional proteins and generate the total protein fraction. The remaining lysate was centrifuged for 20 minutes at 10,000 g in pre-weighed microcentrifuge tubes. Supernatant was removed and added to an equal volume of 1% Triton X-100 buffer containing 8 M urea and 2 M thiourea for the soluble protein fraction. Pellets were weighed and suspended in 1% Triton X-100 buffer containing 4 M urea and 1 M thiourea to a final concentration of 30 mg/ml. A motorized pestle was used to dissolve the pellet and generate the insoluble protein fraction. NuPAGE sample buffer was added to all fractions prior to sonication and centrifugation for 20 minutes at 10,000 g. DTT was added to a final concentration of 100 mM and samples reduced for 30 minutes at 37°C before subjection to SDS-PAGE electrophoresis and Western blotting, as described above. Negligible differences in extraction of insoluble Cx43 were detected using solubilization buffers containing SDS or with higher concentrations of urea. Once quantified, all values were normalized to α-tubulin, and subsequently soluble and insoluble values were normalized to the appropriate control amounts of Cx43.
Preparation of whole protein lysates from human and mouse heart tissue.
Tissue was homogenized in RIPA buffer, sonicated, and prepared for Western blotting as described above.
EB1-K89E expression for Cx43 surface biotinylation.
pLenti6.3-Cx43 HeLa cells were plated in 100-mm dishes at a concentration of 106 cells/dish and allowed to adhere overnight. Cells were transfected with pcDNA3.1-V5-EB1 or pcDNA3.1-V5-EB1-K89E (5 μg/dish) using Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions. After transfection, the medium was changed and Dynasore added to a final concentration of 80 μM. Biotinylation was performed as described above 36 hours after transfection.
Quantification of Western blotting.
All blots were imaged using the Versadoc 4000 MP (BioRad). Flat-fielding was used, and Quantity One (BioRad) analysis software was used to quantify individual bands. Samples were normalized to GAPDH, α-tubulin, or β-actin. Graphs were plotted and statistics performed using Prism 5 software (GraphPad).
All quantitative data were analyzed using Prism 5 software (GraphPad) and expressed as mean ± SEM. A 2-tailed unpaired Student’s t test was used to analyze data containing 2 groups, and a 1-way ANOVA with Bonferroni post-test was used to analyze data with 3 or more groups. In both cases, a P value less than 0.05 was deemed statistically significant.