Generation of Monoclonal Antibodies Specific for Phosphorylated Forms of Perilipin 1A
To generate novel monoclonal antibodies directed to phosphorylated forms of perilipin 1A, peptides analogous to the amino acid sequences surrounding serine 497 (PKA-site 5) or serine 522 (PKA-site 6) of human perilipin 1A were synthesized. The serines of these peptides were substituted with a chemical moiety (see Materials and Methods), which acts as a mimetic for phospho-serine. Mice were injected with the peptides, and sera-positive animals were screened for MAb-secreting hybridomas. Aliquots of media from the hybridomas were tested against the inoculating peptides via ELISA. Clones that scored positive for recognition of the inoculating peptides by ELISA were evaluated for their ability to produce antibodies that visually label adipocytes pretreated with forskolin in a pattern consistent with perilipin 1A. We selected two monoclonal antibodies, designated anti-pPeri-site 5 and anti-pPeri-site 6, that showed strong ELISA reactions and appropriate labeling patterns.
Validation of the Specificity of the Antibodies
To evaluate the specificity of the anti-pPeri-site 5 and anti-pPeri-site 6 antibodies, perilipin 1A mutant plasmids were utilized, in which alanine was substituted for either serine 497 (to remove PKA-site 5) or serine 522 (to remove PKA-site 6). The coding region of each perilipin 1A mutant construct was fused to mCherry, a red channel fluorescent protein, to create mCh-perilipin S497A and mCh-perilipin S522A. Separate HeLa cell preparations were transiently transfected with GFP-perilipin 1A (wt), mCh-perilipin 1A (S497A), or mCh-perilipin 1A (S522A). The cells were then exposed to oleic acid to promote formation of lipid droplets for 24 h. Subsequently, FSK+IBMX was used to activate PKA. Cells were then fixed, stained for nuclei, and labeled with either anti-pPeri-site 5 or anti-pPeri-site 6. Antibody labeling was visualized with secondary anti-mouse antibodies conjugated to a far-red fluorophore.
For cells prepared in this manner, the anti-pPeri-site 5 antibody labeled cells expressing wild type perilipin 1A (), but did not label cells expressing perilipin 1A with the S497A mutation (). Similarly, the anti-pPeri-site 6 antibody also labeled cells expressing wild type perilipin 1A (), but did not label cells expressing perilipin 1A with the S522A mutation (). These experiments demonstrate that specific labeling of perilipin 1A by anti-pPeri-site 5 and anti-pPeri-site 6 requires serine phosphorylation at position 497 and 522, respectively.
Determination of antibody specificity for perilipin 1A PKA-site 5, and PKA-site 6.
Guinea pig polyclonal antibody #GP29, raised against the N-terminus of perilipin 1A, is widely used to label human 
and mouse 3T3L1 adipocytes 
. To further validate the labeling of perilipin 1A by the anti-pPeri-site 5 and anti-pPeri-site 6 antibodies, 3T3L1 adipocytes were exposed to either 6 µM FSK or 100 nM L-γ-MSH for 20 minutes, then fixed, permeabilized, and colabeled with either anti-pPeri-site 5 plus GP29, or with anti-pPeri-site 6 plus GP29. This was possible since the murine and guinea pig antibodies are from different host animals, and can thus be specifically visualized with different secondary antibodies and fluorophores.
For images acquired from these FSK-treated adipocytes, via conventional microscopy, the labeling by anti-pPeri-site 5 was virtually identical to the labeling pattern obtained with GP29 ( vs. 3B). Furthermore, virtually identical labeling patterns were also seen for anti-pPeri-site 6 compared to GP29 ( vs. 3D). Indeed, Pearsons’ Correlation Coefficients, calculated on a cell by cell basis by CyteSeer®, yielded a value of 0.96 for the cell depicted in . For 3C and 3D, there are two juxtaposed cells (note the two dark central regions in 3D, which correspond to the location of separate nuclei), for which CyteSeer® calculated Pr values of 0.88 (cell 1) and 0.94 (cell 2), respectively. Pr has a theoretical range from −1.0 (perfect exclusion) to +1.0 (perfect coincidence). Thus, the observed Pr values strongly support the observation from visual inspection that distribution of phospho-perilipin 1A labeled by the monoclonal antibodies coincides with the distribution of total perilipin 1A labeled by GP29. For all of the antibodies, labeling was specific for the edges of lipid droplets, which is the known intracellular location of perilipin 1A. Virtually identical images for anti-pPeri-site 5 and GP29 and for anti-pPeri-site 6 and GP29 were also obtained by confocal microscopy (), confirming the labeling at the edges of the lipid droplets by both anti-pPeri-site 5 and anti-pPeri-site 6, and the coincidence of the labeling with GP29.
Labeling of adipocytes with anti-pPeri-site 5, anti-pPeri-site 6, and GP29.
In a further experiment to test the specificity of the antibodies, human preadipocytes were transfected with either control siRNA (scrambled sequence) or an siRNA corresponding to human perilipin 1A, at concentrations ranging from 0 to 50 nM. Following transfection, the cells were exposed to Differentiation Medium for 6 days, treated with 6 µM FSK for 20 minutes, and fixed and labeled for nuclei, lipid droplets, and for both phospho-perilipin (utilizing either anti-pPeri-site 5 or anti-pPeri-site 6) and total perilipin (utilizing GP29). For cells transfected with 10 nM control siRNA, most of the cells featured numerous lipid droplets and were strongly labeled by GP29, anti-pPeri-site 5, and anti-pPeri-site 6 (). In contrast, cells transfected with siRNA to perilipin 1A featured much weaker labeling by anti-pPeri-site-5, anti-pPeri-site6, and GP29. Interestingly, the cells transfected with the perilipin siRNA also featured fewer lipid droplets. This is consistent with expectations, as knockout of perilipin 1A in mice results in a leaner phenotype 
and siRNA to perilipin also downregulates lipid droplets in a murine adipocyte cell model 
. The potential explanation for this is that reduction of perilipin expression in adipocytes likely leads to increased basal lipolysis and reduction of triglyceride stores.
Downregulation of anti-pPeri-site 5, anti-pPeri-site 6, and GP29 labeling by siRNA to perilipin 1A.
To quantify the effects of the siRNAs, images from the experiment were analyzed with CyteSeer®. Area Pm values were not affected by the control siRNA, but the perilipin siRNA reduced labeling by GP29, anti-pPeri-site5, and anti-pPeri-site 6 in a very similar fashion, up to 90% ().
The above results confirm that anti-pPeri-site 5 labels perilipin 1A which has been phosphorylated at PKA-site 5, whereas anti-pPeri-site 6 labels perilipin 1A, which has been phosphorylated at PKA-site 6. Note that for the purposes of quantification, perilipin 1A labeled by the anti-phospho-perilipin 1A antibodies, will be referred to as pPeri-site 5 and pPeri-site 6, respectively.
Labeling of Endogenous Phospho-perilipin 1A in Human Adipocytes at the Initiation of Lipolysis
Next, experiments were conducted to test the ability of these antibodies to recognize endogenous phospho-perilipin 1A in adipocytes under lipolytic treatments. Human adipocytes exposed to control medium showed little labeling for with the anti-pPeri-site 5 antibody (). In contrast, following exposure to FSK/IBMX, labeling by anti-pPeri-site 5 was very strongly increased (), suggesting FSK/IBMX strongly increases phosphorylation of perilipin 1A at PKA-site 5. Similarly, for anti-pPeri-site 6, there was minimal labeling of human adipocytes under basal conditions (), and prominent labeling following exposure to FSK/IBMX (), suggesting that FSK/IBMX also leads to phosphorylation of perilipin 1A PKA-site 6.
Labeling of FSK-treated human adipocytes with anti-pPeri-site 5 and anti-pPeri-site 6 antibodies.
Differential Phosphorylation of Perilipin 1A in Response to L-γ-MSH Versus FSK
and others 
have examined the phosphorylation of HSL in 3T3L1 adipocytes in response to FSK and L-γ-MSH. To examine the phosphorylation of perilipin 1A in response to these agents, murine 3T3L1 adipocytes were exposed to either FSK or L-γ-MSH for 5 minutes, then fixed, and labeled for nuclei and lipid droplets, along with either anti-pPeri-site 5 or anti-pPeri-site 6. 3T3L1 adipocytes exposed to control medium exhibited relatively little labeling with anti-pPeri-site 5 (). Treating the cells with FSK (6 µM) or L-γ-MSH (100 nM) led to strong and similar increases in labeling for anti-pPeri-site 5 (). Interestingly, the results obtained with the anti-pPeri-site 6 antibody were qualitatively distinct from the results for anti-pPeri-site 5. For the anti-pPeri-site 6 antibody, labeling was dim for control cells () and increased by both FSK () and L-γ-MSH (); however, the effect of L-γ-MSH was stronger than the effect of FSK.
Differential phosphorylation of perilipin 1A by forskolin (FSK) and LγMSH.
To quantify the agonist-induced appearances of pPeri-site 5 and pPeri-site 6, images from the experiment were analyzed with CyteSeer® utilizing the Colocalization Algorithm. For pPeri-site 5, Area Pm (the area of pixels that are positive for pPeri-site 5 on a per cell basis) averaged 27 µm2/cell for control cells (), suggesting a small but measurable degree of basal phosphorylation of perilipin 1A at PKA-site 5. Exposure to FSK or L-γ-MSH led to 7-fold and 8-fold increases in Area Pm, respectively. These responses were significantly different compared to control wells (p<0.01), but were not significantly different from each other. For pPeri-site 6, Area Pm values for controls averaged 7 µm2/cell (). Exposure to FSK or L-γ-MSH led to 11-fold and 27-fold increases in Area Pm, respectively. Statistically, these responses were different both from the controls and from each other at a high level of significance (p<0.001). Thus, in this experiment, L-γ-MSH was nearly 3-fold more effective than FSK at inducing the appearance of pPeri-site 6.
Concurrent Analysis of Phospho-perilipin 1A and Phospho-HSL in 3T3L1 Adipocytes Subjected to FSK or L-γ-MSH
In the experiment described above, FSK and L-γ-MSH were equally effective at inducing the appearance of pPeri-site 5, but L-γ-MSH was a stronger agonist at inducing the appearance of pPeri-site 6. This prompted us to ask whether or not these agents might also lead to differential phosphorylation of HSL. Accordingly, a time course experiment was conducted in which 3T3L1 adipocytes were exposed to control medium, 6 µM FSK, or 200 nM L-γ-MSH, for time periods of 1, 5, or 20 minutes. The cells were then fixed as before, but co-labeled for both phospho-perilipin 1A and phospho-HSL. This was possible since the anti-perilipin 1A antibodies were raised in mice, whereas the anti HSL antibodies were raised in rabbit, and thus these antibodies are immunologically distinct. In the experiments shown, adipocytes were co-labeled with anti-pPeri-site 5 plus anti-pHSL-serine 563 antibodies, or co-labeled for with anti-pPeri-site 6 plus anti-pHSL-serine 660 antibodies. Following labeling, the cells were visualized in 4 fluorescent channels and the images analyzed with the CyteSeer® Colocalization algorithm.
For pPeri-site 5 (), Area Pm values averaged about 20 µm2 for control cells. Exposure to either FSK or L-γ-MSH led to approximately 4-fold increases in Area Pm which was immediate and sustained throughout the time course. Thus, phosphorylation of perilipin 1A at PKA-site 5 occurs rapidly for both agonists, and, FSK and L-γ-MSH were equally effective.
Phosphorylation of Perilipin 1A and HSL in response to FSK and L-γ-MSH.
For pHSL-serine 563 (), Area Pm values for controls were negligible. For cells exposed to FSK for 1 minute, Area Pm for FSK was approx. 4 µm2 whereas Area Pm for L-γ-MSH averaged about 20 µm2, which was significantly increased relative to the controls. At the 5 minute time point, Area Pm values averaged approx. 19 µm2 for FSK and about 41 µm2 for L-γ-MSH and these values were statistically different from controls. At the 20 minute time point, the data for FSK and L-γ-MSH were somewhat higher than for the 5 minute time point. At all time points, the differences between the L-γ-MSH and FSK were statistically significant.
For pPeri-site 6 () at the one minute time point, Area Pm values averaged about 22 µm2 for control samples, and FSK and L-γ-MSH increased Area Pm by approximate 20% and 50%, respectively. At the 5 minute time point, the response for L-γ-MSH was 2-fold greater than the basal level of Area Pm, while the effect of FSK was not significantly different from control cells. At the 20 minute time point, FSK and L-γ-MSH increased Area Pm by approximately 3-fold and 5-fold, respectively. At all time points, the data values obtained with L-γ-MSH were significantly elevated compared to the data for FSK.
For pHSL-serine 660 (), Area Pm values averaged about 5 µm2 for control cells at the 1 minute time point, whereas FSK and L-γ-MSH increased Area Pm by approximately 4-fold and 9-fold, respectively. At the 5 minute time point, FSK and L-γ-MSH elicited similar responses and exhibited levels approximately 10-fold above the control values. At the 20 minute time point, both agents elicited very strong, virtually identical responses.
It is worth emphasizing that the data for were obtained from the same wells on the 96-well dish. Thus, FSK and L-γ-MSH elicited the appearance of pPeri-site 5 and pHSL-serine 563 with different kinetics and agonist efficacies within the same samples. Similarly, the data for were also obtained from the same wells on the 96-well dish, so FSK and L-γ-MSH also elicited the appearance of pPeri-site 6 and pHSL-serine 660 with different kinetics and different agonist efficacies within the same samples. There are notable similarities in the data obtained for pPeri-site 5 and pHSL-serine 660, including more rapid phosphorylation of these sites, and equal efficacies of FSK and L-γ-MSH at the 5 and 20 minute time points, and similarities between the data obtained for pPeri-site 6 and pHSL-serine 563 (phosphorylation of these sites occurred more slowly, and L-γ-MSH was a stronger agonist than FSK). Results with similar kinetics and relative magnitudes of responses to FSK and L-γ-MSH were also obtained in two additional time course experiments.
Differential Phosphorylation of Perilipin and HSL in Response to a Panel of Lipolytic Agents
In related experiments, 3T3L1 cells were exposed to a panel of lipolytic agents, which consisted of 0.01 µM isoproterenol, 0.1 µM isoproterenol, 1 µM FSK, 6 µM FSK, and 0.2 µM L-γ-MSH, for 20 minutes, then fixed and labeled in a manner identical to the experiments described above.
For pPeri-site 5 (), Area Pm for control cells averaged approximately 70 µm2. The low and high concentrations of isoproterenol increased Area Pm by approximately 30%, or 50%, respectively. The results for both concentrations of FSK, and for L-γ-MSH were similar, with each agent increasing Area Pm by approximately 2.5-fold.
Differential phosphorylation of perilipin 1A and HSL in response to a panel of lipolytic agents.
For pHSL-serine 563 (), there was negligible labeling of the control cells. The low and high concentrations of isoproterenol increased Area Pm to approximately 20 µm2 and 42 µm2; both of these values were significantly different from controls and from each other. For FSK, the lower concentration increased Area Pm to about 40 µm2 whereas the high dose increased Area Pm to about 80 µm2. L-γ-MSH increased Area Pm to approximately 103 µm2, which was the greatest observed effect of the treatments, and the effect was significantly increased relative to the data for the high dose of FSK.
For pPeri-site 6 (), Area Pm averaged 25 µm2 for control cells. Exposure to the low and high concentrations of isoproterenol increased Area Pm by 2-fold and 3-fold, respectively. The low and high concentrations of FSK elicited approximately 2.5-fold and 3-fold increases in Area Pm. L-γ-MSH increased Area Pm approx. 5-fold, which was the strongest response observed in these experiments.
For pHSL-serine 660 (), Area Pm averaged 48 µm2 for controls. Exposure to the low and high doses of isoproterenol increased Area Pm by 70% and 2-fold, respectively. For FSK, the low and high concentrations elicited 3.8-fold and 4.3-fold increases in Area Pm, whereas L-γ-MSH increased Area PM by 4.2-fold.
In the above experiment, the response patterns obtained for pPeri-site 5 and pHSL-serine 660 are strikingly similar with perfect agreement in the rank order effectiveness of the treatments. The response patterns for pPeri-site 6 and pHSL-serine 563 are also very similar to each other and are different from the response patterns for pPeri-site 5 and pHSL-serine 660.
Experiments with Blocking Peptides
The close similarities between the results for pPeri-site 5 and for pHSL-serine660, and the similarity between the results for pPeri-site 6 and pHSL-serine 563 raised the possibility that the antibodies to phospho-perilipin may be labeling phospho-HSL, or vice versa. To test if these peptides would block labeling of the cells by the primary antibodies, cells were incubated with primary antibodies that were pre-incubated with blocking peptides corresponding to pPeri site 5, pPeri site 6, or pHSL serine 660.
For 3T3L1 adipocytes treated with 1 µM isoproterenol, preincubation with the pPeri site 5 peptide reduced the overall image intensities resulting from labeling by the anti-pPeri site 5 antibody, and the Tii Pi Pm data parameter (which represents the protein labeling intensity, quantified on a per cell basis) (). Thus, binding of anti-pPeri site 5 antibody to its target is blocked by peptide corresponding to pPeri site 5, confirming the specificity of the antibody. In contrast, the pPeri site 5 peptide did not reduce image intensities or Tii Pi Pm resulting from labeling by the anti-pHSL serine 660 antibody (); thus, the anti-pHSL serine 660 antibody does not bind to pPeri site 5.
Tests for antibody specificity utilizing blocking peptides.
In a reciprocal experiment, also featuring 3T3L1 adipocytes treated with 1 µM isoproterenol, preincubation of anti-pPeri-site 5 antibody with the pHSLser660 peptide did not reduce overall image intensities or Tii Pi Pm obtained with the anti-pPeri-site 5 antibody (); in contrast, the pHSL serine 660 peptide strongly reduced the image intensities and Tii Pi Pm values obtained with the anti-pHSL-serine 660 antibody (). The results demonstrate that the anti-pPeri-site 5 antibody likely does not bind to pHSL serine 660 and confirms the specificity of the anti-pHSL-serine 660 antibody.
Finally, for 3T3L1 adipocytes treated with 100 nM L-γ-MSH, preincubation of anti-pPeri-site 6 antibody with a peptide corresponding to pPeri-site 6 strongly reduced image intensities and the Tii Pi Pm values obtained with anti-pPeri-site 6 antibody, but did not reduce overall image intensities or the Tii Pi Pm values obtained with the anti-pHSL-serine 563 antibody (). The results confirm the specificity of the anti-pPeri-site 6 antibody for pPeri-site 6, and demonstrate that the anti-pHSL-serine 563 antibody does not bind to pPeri-site 6.
Overall, the results with the blocking peptides confirmed the specificity of the phospho-perilipin and phospho-HSL antibodies for labeling their intended targets and indicate it is highly unlikely that the anti-perilipin 1A antibodies recognize HSL, or vice versa.
Analysis of Perilipin 1A Phosphorylation via Western Blotting
To explore additional applications for the monoclonal antibodies against phospho-perilipin, we tested the ability of these antibodies to identify phospho-perilipin 1A by Western blotting. In our hands, anti-pPeri-site 5 did not visualize specific bands on Western blots. In contrast, anti-pPeri-site 6 labeled a single band, which migrated at approx. 60 KDa, which is consistent with the molecular weight of perilipin 1A, and the intensity of this band was strongly increased in samples for 3T3L1 cells treated with forskolin (). Furthermore, for experiments in which adipocytes were treated for 5 minutes with control, 6 µM FSK, 1 µM isoproterenol, and 100 nM L-γ-MSH, the bands from cells treated with isoproterenol and L-γ-MSH recognized by the anti-pPeri-site 6 antibody were more intense than the band for cells treated with FSK (). The observation is consistent with the previous observations that L-γ-MSH is a stronger agonist than FSK for phosphorylation of perilipin at PKA-site 6, as quantified via the microscopy methods. The strong response to isoproterenol, vs. FSK is somewhat unexpected considering the results from the microscopy assay. However, in the experiment for the Western blot, the cells were treated with a higher concentration of isoproterenol than utilized in the microscopy experiment. Results similar to those shown in were obtained in 2 additional experiments with anti-pPeri-site 6. In all, the results confirm the anti-pPeri-site 6 antibody recognizes phosphorylated perilipin 1A and this antibody is compatible with Western blotting.
Phospho-perilipin 1A visualized via Western blotting.