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1.
Figure 10

Figure 10. Phospho-perilipin 1A visualized via Western blotting.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

3T3L1 adipocytes were exposed to lipolytic agonist for 5 minutes; whole cell lysates were then prepared and subjected to SDS-PAGE/Western blotting (20 µg protein/lane) utilizing the anti-pPeri-site 6 antibody. A, Results are shown for cells exposed to either control medium (C) or 6 µM FSK (F). B, Results are shown for a separate experiment in which cells were exposed to control medium, 6 µM FSK, 1 µM isoproterenol (I), or 100 nM L-γ-MSH (M).

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
2.
Figure 5

Figure 5. Labeling of FSK-treated human adipocytes with anti-pPeri-site 5 and anti-pPeri-site 6 antibodies.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

Human subcutaneous adipocytes were exposed to either control medium, or medium supplemented with 10 µM FSK and 500 µM IBMX for 2 minutes, then fixed and labeled for nuclei, lipid droplets, and with either anti-pPeri-site 5 or anti-pPeri-site 6. A goat-anti-mouse secondary antibody coupled to Texas Red was used to visualize the phospho-perilipin antibodies in the red fluorescent channel. A, Control adipocytes are shown visualized for nuclei (blue) and lipid droplets (green). B, the same field is shown visualized for anti-pPeri-site 5 (red). C, FSK/IBMX-treated adipocytes are shown visualized for nuclei and lipid droplets. D, The same field is shown visualized for anti-pPeri-site 5. E, Control adipocytes are shown visualized for nuclei and lipid droplets. F, The same field is shown visualized for anti-pPeri-site 6. G, FSK/IBMX-treated adipocytes are shown visualized for nuclei and lipid droplets. H, The same field is shown, visualized for anti-pPeri-site 6. Scale bar = 50 µm.

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
3.
Figure 2

Figure 2. Determination of antibody specificity for perilipin 1A PKA-site 5, and PKA-site 6.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

Hela cells were transfected separately with plasmids encoding wild-type (GFP, green) or mutant (mCherry, red) perilipin 1A (PLIN1) plasmids. Oleic acid was added for 24 h, followed by 7 min treatment with 24 µM forskolin plus 125 µM IBMX and the cells labeled with either anti-pPeri-site 5 or anti-pPeri-site 6. For each condition, images are shown for nuclei (DAPI), fluorescent protein (either GFP or m-Cherry), or for antibody labeling (the far red fluorescence channel). A, Cells expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 5. B, Cells expressing mCh-perilipin 1A S497A, which did not label for anti-pPeri-site 5. C, Cells expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 6. D, Cells expressing mCh-perilipin 1A S522A, which did not label with anti-pPeri-site 6.

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
4.
Figure 1

Figure 1. Regulation of lipolysis in adipocytes.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

A, A current model for the hormonal regulation of lipolysis initiation is shown. Proteins depicted include perilipin 1A (Peri), Hormone Sensitive Lipase (HSL), Adipocyte Triglyceride Lipase (ATGL), CGI-58, and PKA. Lipid species depicted include triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), and fatty acid (FA). Under basal conditions, perilipin and HSL are unphosphorylated and HSL is found throughout the cytoplasm. Stimulation of lipolysis involves activation of PKA, phosphorylation of perilipin 1A and HSL, release of CGI-58 from perilipin, binding of CGI-58 to ATGL, and translocation of HSL to perilipin. TAG is sequentially processed to DAG by ATGL and to MAG by HSL with FA released at each step. B, Amino acid sequences are shown for perilipin 1A PKA site 5, and PKA site 6, and for HSL serine 563 and serine 660. The target serine in each sequence is underlined.

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
5.
Figure 4

Figure 4. Downregulation of anti-pPeri-site 5, anti-pPeri-site 6, and GP29 labeling by siRNA to perilipin 1A.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

Preadipocytes were transfected with either control or perilipin 1A siRNA (0 to 50 nM), exposed to differentiation medium for 6 days, treated with 6 µM FSK for 20 minutes, then fixed and labeled for nuclei (blue), lipid (green), and either anti-pPeri-site 5 or anti-pPeri-site 6 (red) plus GP29 (yellow). A, Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 5 plus GP29. B, Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 6 plus GP29. C, D, and E are mean values for Area of the Protein mask (Area Pm), for GP29, anti-pPeri-site 5, and anti-pPeri-site 6, respectively. Data are normalized to the 0 siRNA control, and each bar represents the mean ± SD, for n = 6 wells. *** p<0.001 for perilipin vs. control siRNA at each concentration (Student’s t-test).

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
6.
Figure 6

Figure 6. Differential phosphorylation of perilipin 1A by forskolin (FSK) and LγMSH.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

3T3L1 adipocytes were exposed to either FSK (6 µM) or L-γ-MSH (100 nM) for 5 minutes, then fixed and labeled for nuclei (blue), lipid droplets (green), and phosphorylated perilipin (red). The cells were then imaged (20Xobjective, 4 images/well) and the images analyzed utilizing the Colocalization algorithm. A, B, and C, Images are shown of adipocytes exposed to control, FSK, or LγMSH, respectively, labeled with anti-pPeri-site 5. D, E, and F, Images are shown of adipocytes exposed to control, FSK, LγMSH, respectively, labeled with anti-pPeri-site 6. G and H represent Area Pm for pPeri-site 5 and for pPeri-site 6, respectively. Each bar represents the mean SD for n = 3 wells/condition (an average of 532 cells/well) for G, or n = 8 well/condition (an average of 272 cells/well) for H. ** p<0.01 vs. Con (ANOVA followed by Tukey’s test). *** P<0.001 vs. Con. ### p<0.001 vs. FSK. Scale bar = 50 µm.

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
7.
Figure 7

Figure 7. Phosphorylation of Perilipin 1A and HSL in response to FSK and L-γ-MSH.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

3T3L1 adipocytes were exposed to control medium, 6 µM forskolin (F), or 200 nM L-γ-MSH (M) for 1, 5, or 20 minutes, then fixed and co-labeled with either anti-pPeri-site 5+ anti-pHSL-serine 563, or anti-pPeri-site 6+ anti-pHSL-serine 660. Images were obtained with a 20X objective (4 images/well, representing an average of 930 cells/well). A and B represent Area Pm values obtained for pPeri-site 5 and pHSL-serine 563. C and D represent Area Pm values obtained for pPeri-site 6 and pHSL-serine 660, respectively. Each bar represents the mean ± SD for n = 7 to 8 wells. ** p<0.01 compared to controls (ANOVA followed by Tukey’s test); *** p<0.001 compared to controls. a p<0.05 vs. controls and M; b p<0.05 vs. controls and F.

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
8.
Figure 8

Figure 8. Differential phosphorylation of perilipin 1A and HSL in response to a panel of lipolytic agents.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

3T3L1 adipocytes were exposed to the indicated concentrations (µM) of isoproterenol (ISO), FSK, and L-γ-MSH for 20 minutes, then fixed and labeled either PKA-site 5+ pHSL-serine 563 or PKA-site 6+ pHSL-serine 660. The cells were imaged with a 20X objective, 4 images/well, yielding an average of 360 cells/well. Area Pm values are shown for (A) PKA-site 5 (B), pHSL-serine 563, (C) PKA-site 6, and (D), pHSL-serine 660, respectively. Each bar represents the mean ± SD for n = 8 wells. * p<0.05 vs. control (ANOVA followed by Tukey’s test procedure); *** P<0.001 vs. controls. Within each panel, conditions distinct from one another at the p<0.05 level are also designated (a,b,c,d).

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
9.
Figure 9

Figure 9. Tests for antibody specificity utilizing blocking peptides.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

3T3L1 adipocytes were exposed to either 1 µM isoproterenol or 100 nM L-γ-MSH. Prior to labeling, primary antibodies were preincubated with the indicated amounts (µg) of blocking peptides corresponding to pPeri-site 5, pPeri-site 6, or pHSL-serine 660. Anti-phospho-perilipin 1A and anti-phospho-HSL antibodies were visualized in the red and far-red fluorescence channels, respectively. A, Results are shown for cells treated with isoproterenol for 15 minutes in which anti-pPeri-site 5 and anti-pHSLserine 660 were blocked with pPeri-site 5. Upper panels depict cell images. Lower bar graphs depict Tii Pi Pm data for pPeri-site 5 and pHSL-serine 660. B, Results are shown for cells treated with isoproterenol for 10 minutes in which anti-pPeri-site 5 and anti-pHSL-serine 660 were blocked with pHSL-serine 660. C, Results are shown cells treated with L-γ-MSH for 7 minutes in which anti-pPeri-site 6 and anti-pHSLserine563 were blocked with pPeri-site 6. For A, each bar represents a single well; for B and C, each bar represents the mean ± SD for n = 3 wells.

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.
10.
Figure 3

Figure 3. Labeling of adipocytes with anti-pPeri-site 5, anti-pPeri-site 6, and GP29.. From: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis.

3T3L1 adipocytes were exposed to agonists (either 6 µM FSK or 100 nM L-γ-MSH) for 20 minutes, then fixed, and labeled with either anti-pPeri-site 5 or anti-pPeri-site 6 and GP29. Conventional microscopy (A–D). Alexa Fluor® 488 goat-anti-mouse and Alexa Fluor® 647 goat anti-guinea pig secondary antibodies were used to visualize the mouse monoclonal and guinea pig polyclonal antibodies, respectively. A, A FSK-treated cell visualized for anti-pPeri-site 5. B, The same cell, visualized for GP29. C, A cluster of two FSK-treated cells (labeled 1 and 2) visualized for anti-pPeri site 6; the dashed line is the boundary between the cells as estimated by CyteSeer®. D, The same cell cluster, visualized for GP29. Images were acquired with a 40×0.75 NA objective. E–H, Confocal microscopy. Alexa Fluor® 568 goat-anti-mouse and Alexa Fluor® 647 goat anti-guinea pig secondary antibodies were used to visualize the mouse monoclonal and guinea pig polyclonal antibody labels, respectively. E, FSK-treated cells visualized for anti-pPeri-site 5. F, The same cells visualized for GP29. Images were acquired with a 20×0.8 NA objective. G, L-γ-MSH-treated cells visualized for anti-pPeri-site 6. H, The same cells visualized for GP29. Images were acquired with a 63×1.4 NA objective. Examples of prominent labeling at the edges of lipid droplets are indicated by arrows (lipid droplets, which were visualized in a separate channel, are not shown). Scale bars are 50 µm.

Patrick M. McDonough, et al. PLoS One. 2013;8(2):e55511.

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