Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: (A) Radioligand binding assay for detection of anti-PLIN1 antibodies. Radiolabeled PLIN1 protein was incubated with sera from healthy controls (HC, n = 54) or from APS1 patients with or without lipodystrophy (APS1 +/- LD, n = 1/68). Dotted line indicates mean + 3SD of healthy controls. (B) Validation of autoantibodies to PLIN1 in orthogonal cell-based assay. Fixed stomach tissue of mice was mounted and immunostained with sera from Case report patient 1 and commercial antibody to PLIN1. A mix of secondary antibodies anti-Human IgG-alexa547 and anti-Rabbit alexa488 at 1:2000 were used to visualize Human IgG and PLIN1 antibodies, respectively. A Merge is provided on the right of individual images. Images taken at 20X magnification. Note yellow in Merge indicating colocalization of PLIN1 (Green) and Human IgG (RED). DAPI is blue and indicates nuclei. Scale bar indicates 100 microns.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Radio Ligand Binding Assay, Incubation, Cell Based Assay

Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: Autoantibodies to PLIN1 in sera from a patient with autoimmune AGL following cancer immunotherapy. (A) Radioligand binding assays screening for PLIN1 antibodies in sera from all time points from index patient 2 as well as checkpoint-treated control patients without lipodystrophy (n=7), as done for case report 1. Dotted line indicates mean + 3 std. dev. of healthy controls (n = 11). Numbers to the right of circles indicate the time point series, for reference in panel B. The left panel describes the clinical timeline corresponding to timepoints T0-T4. CPI was discontinued at 34 cycles (16 months) due to progressive weight loss and elevated LFTs, with futher work-up over subsequent months. (B) Validation of autoantibodies to PLIN1 using immunohistochemistry on mouse enteric tissue, as done in . Sera was used from either a control (immunotherapy but no AGL) or case report 2 (Immunotherapy with AGL) with various time points. Each column represents an individual sample, from left to right ; sera from checkpoint control pre-treatment, sera from checkpoint control post treatment with no autoimmunity, Case report 1 pre-therapy, Case report 1 post-therapy-1, Case report 1 post-therapy-3, Case report 1 post-therapy-4. Images are 600×600 pixel insets from original 40X image. Scale bar represents 100 microns. CP = Checkpoint patient controls.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Binding Assay, Immunohistochemistry

Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: Discovery and validation of autoantibodies to Perilipin-1 in Aire -/- mouse sera. (A) PhIP-Seq analysis in Aire -/- (“KO”) and Aire +/+ (“WT”) mice. Aggregated Plin1 PhIP-Seq data from Aire -/- (KO, n=4) and Aire +/+ (WT, n = 3) mice. (B) Whole cell lysates generated from 293T cells expressing full-length mouse PLIN1 were incubated with sera from Aire -/- (KO, n=4) or Aire +/+ (WT, n=3) mice. Antibodies were immunoprecipitated using AG beads and IP elutions were subject to SDS-PAGE immunoblotting. AG lane indicates an IP using AG beads only, no sera. Input lane indicates loading of whole cell lysate with and without (-) transfection of PLIN1-myc-flag plasmid. IP elutions and Input were immunostained with either anti-Flag IgG to identify positive anti-Plin1 signal, or anti-mouse IgG to show qualitative capture of IgG from sera. (C) Representative image of immunohistochemistry on mouse enteric tissue showing positive colocalization of antibodies from Aire -/- sera (KO) and commercial anti-Plin1 IgG. Primary antibodies from Aire -/- mouse (KO) sera and commercial antibody to PLIN1 were visualized with secondaries to Mouse IgG (Alexa-567, RED) and Rabbit IgG (Alexa-488, GREEN), respectively. DAPI (BLUE) stains nuclei.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Generated, Expressing, Incubation, Immunoprecipitation, SDS Page, Western Blot, Transfection, Plasmid Preparation, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Autoantibodies Against Perilipin 1 as a Cause of Acquired Generalized Lipodystrophy

doi: 10.3389/fimmu.2018.02142

Figure Lengend Snippet: Candidate antigens in acquired generalized lipodystrophy.

Article Snippet: ELISA microtiter plates (Medisorb, Nunc, VWR) were coated with 100 ng/well of recombinant PLIN1 (OriGene) in carbonate-bicarbonate buffer pH 9.6 (overnight, 4°C).

Techniques: RNA Expression, Molecular Weight, Binding Assay, Clinical Proteomics, Membrane

Journal: Frontiers in Immunology

Article Title: Autoantibodies Against Perilipin 1 as a Cause of Acquired Generalized Lipodystrophy

doi: 10.3389/fimmu.2018.02142

Figure Lengend Snippet: Characterization of anti-PLIN1 autoantibodies in patients with AGL. (A) Equal amounts of human adipose tissue were electrophoresed under reducing (2BME +) and nonreducing (2BME –) conditions. Membrane was incubated with either reactive serum samples from two patients with AGL as source of antibodies or a polyclonal anti-PLIN1 IgG (#AF6615) and detected with appropriate secondary antibodies. (B) Results of ELISA analyses demonstrating that IgG from three patients with AGL (ALG1, AGL2, and ALG3) binds to recombinant human PLIN1. Neither serum from healthy donors ( n = 20, NHS), nor from patients with acquired partial lipodystrophy ( n = 8, APL), localized lipoatrophy due to intradermic insulin injections ( n = 11, I-LA), and systemic lupus erythematosus ( n = 10, SLE) reacted against recombinant human PLIN1 on ELISA assay. Samples were run in duplicates. Each point represents the mean value of optical density (O.D.) for each sample. (C) White adipose tissue extracts were blotted with serum samples from three anti-PLIN1-positive patients (AGL1 through AGL3), followed by detection with antibodies specific for each human IgG subclass (1 through 4), and peroxidase conjugated anti-subclass-specific IgG antibodies . (D) Blotted human adipose tissue proteins were revealed using serum from a patient with anti-PLIN1 autoantibodies either pre-incubated (right) or not (left) with recombinant human PLIN1. PLIN1 detection is blocked by the recombinant protein, thus demonstrating specificity.

Article Snippet: ELISA microtiter plates (Medisorb, Nunc, VWR) were coated with 100 ng/well of recombinant PLIN1 (OriGene) in carbonate-bicarbonate buffer pH 9.6 (overnight, 4°C).

Techniques: Membrane, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Autoantibodies Against Perilipin 1 as a Cause of Acquired Generalized Lipodystrophy

doi: 10.3389/fimmu.2018.02142

Figure Lengend Snippet: Colocalization of PLIN1 and anti-PLIN1 autoantibodies from patients with acquired generalized lipodystrophy. Confocal microscopic analysis of human preadipocytes revealed colocalization of PLIN1 and anti-PLIN1 IgG from AGL1 on the lipid droplet surface. However, no colocalization was observed when using serum from AGL4 and AGL5 (negative for anti-PLIN1 autoantibodies). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, blue); IgG binding was detected using FITC-conjugated rabbit anti-human IgG (green); PLIN1 was detected with biotin-labeled rabbit IgG followed by Texas Red-labeled streptavidin (red). Scale bars correspond to 18 μm.

Article Snippet: ELISA microtiter plates (Medisorb, Nunc, VWR) were coated with 100 ng/well of recombinant PLIN1 (OriGene) in carbonate-bicarbonate buffer pH 9.6 (overnight, 4°C).

Techniques: Staining, Binding Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Autoantibodies Against Perilipin 1 as a Cause of Acquired Generalized Lipodystrophy

doi: 10.3389/fimmu.2018.02142

Figure Lengend Snippet: Strategy used for the characterization of the domains recognized by anti-PLIN1 autoantibodies. Results of a western blot screening performed on human adipose tissue extracts, under reducing (2BME +) or non-reducing (2BME –) conditions, incubated with serum sample from one patient with acquired generalized lipodystrophy (AGL1), or two polyclonal anti-PLIN1 antibodies directed against different regions of the protein. PαPLIN1 corresponds to antibody #AF6615 from R&D. D418 corresponds to the results obtained using a rabbit polyclonal anti-PLIN1 antibody targeting the C-terminal domain. Arrows indicate unspecific binding by the secondary antibody.

Article Snippet: ELISA microtiter plates (Medisorb, Nunc, VWR) were coated with 100 ng/well of recombinant PLIN1 (OriGene) in carbonate-bicarbonate buffer pH 9.6 (overnight, 4°C).

Techniques: Western Blot, Incubation, Binding Assay

Journal: Frontiers in Immunology

Article Title: Autoantibodies Against Perilipin 1 as a Cause of Acquired Generalized Lipodystrophy

doi: 10.3389/fimmu.2018.02142

Figure Lengend Snippet: Mouse PLIN1 detection using serum from patients with AGL. (A) Tissue extracts were blotted with sera (1:100 dilutions) from patients with AGL known to be reactive (AGL1 and AGL2) or non-reactive (AGL5) with human PLIN1. Reactive sera from patients with AGL detect the two major isoforms of mouse PLIN1 (called PLINA and PLINB). As a positive control, the extract was blotted with a rabbit polyclonal anti-PLIN1 antibody. (B) Confocal microscopic analysis of mouse preadipocytes revealed colocalization of PLIN1 and IgG from patient AGL3 on the lipid droplet surface (merged image, in yellow). However, no colocalization was observed when using serum from one healthy donor (NHS). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, blue); IgG binding was detected using FITC-conjugated rabbit anti-human IgG (green); PLIN1 was detected with biotin-labeled rabbit IgG followed by Texas Red-labeled streptavidin (red). Scale bars correspond to 18 μm.

Article Snippet: ELISA microtiter plates (Medisorb, Nunc, VWR) were coated with 100 ng/well of recombinant PLIN1 (OriGene) in carbonate-bicarbonate buffer pH 9.6 (overnight, 4°C).

Techniques: Positive Control, Staining, Binding Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Autoantibodies Against Perilipin 1 as a Cause of Acquired Generalized Lipodystrophy

doi: 10.3389/fimmu.2018.02142

Figure Lengend Snippet: Functional effects of anti-PLIN1 autoantibodies on lipolysis. Preadipocytes (3T3-L1) were incubated overnight with [ 3 H]palmitic acid at 37°C. (A) Results of the radiometric assessment of basal and stimulated lipolysis after 1, 2, and 3 h in preadipocytes treated with 300 micrograms of IgG purified from one patient with acquired generalized lipodystrophy (AGL3) with anti-PLIN1 autoantibodies and a healthy donor (NHS). Cells treated with IgG from patient AGL3 showed a significant increase in [ 3 H]palmitic acid release under basal ( ** P < 0.01 at 2 h; *** P < 0.001 at 1 and 3 h) compared with cells treated with NHS IgG. Data represent mean ± SD, for triplicates of two independents experiments for each sample. Statistical significance was assessed with two-way ANOVA assay. (B ) Radiolabeled palmitic acid release under basal conditions in preadipocytes treated with 300 micrograms of IgG purified from patient's AGL1, AGL2, AGL4 and AGL5 sera and a NHS. A commercial human PLIN1 Antigen Affinity-purified Polyclonal Antibody (10 μg per well) was used as positive control for blocking lipolysis regulation. Under these conditions, IgG purified from patients AGL1 and AGL2 induced a significant increase of basal lipolysis ( ** P < 0.01), comparable to that induced by a polyclonal anti-PLIN1 antibody (#AF6615 from R&D). Data represent mean ± SD, for triplicates of two independents experiments for each sample. Statistical significance was assessed with one-way ANOVA assay comparing the mean of each column with the mean of a control column (IgG NHS). n.s., not significant.

Article Snippet: ELISA microtiter plates (Medisorb, Nunc, VWR) were coated with 100 ng/well of recombinant PLIN1 (OriGene) in carbonate-bicarbonate buffer pH 9.6 (overnight, 4°C).

Techniques: Functional Assay, Incubation, Purification, Affinity Purification, Positive Control, Blocking Assay, Control

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: 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.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Translocation Assay, Sequencing

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: 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.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Transfection, Mutagenesis, Labeling, Antibody Labeling, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: 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).

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Transfection, Control, Labeling, Concentration Assay

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: 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.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Control, Labeling

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: 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.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Labeling, Control

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: 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.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Labeling, Blocking Assay, Fluorescence

Journal: Autoimmunity

Article Title: Anti-perilipin-1 autoantibodies in autoimmune Addison's disease and related endocrine disorders.

doi: 10.1080/08916934.2025.2461703

Figure Lengend Snippet: Figure 1. Anti-PLIN1-autoAbs in patients with autoimmune endocrine diseases. Anti-PLIN 1-autoAbs indices in APS-1, AAD/APS-2, IAD, GD, T1D, POI and blood donor groups. Mean index values for each patient group are marked by horizontal lines. Threshold for positivity (horizontal dashed red line) has been calculated for the PLIN1 autoantigen based on the mean of indices of blood donors + 3SD (index ≤122). Thirteen dominant negative mutations in AIRE patients (gray dots) are included in the APS-1 patient subgroup. APS-1: autoimmune polyendocrine syndrome type 1; AAD: autoimmune Addison’s disease; APS-2: autoimmune poly endocrine syndrome type 2; IAD: idiopathic Addison’s disease; GD: graves’ disease; T1D: type 1 diabetes; POI: primary ovarian insufficiency; SS: Sjögren’s syndrome.

Article Snippet: For coating of wells, 50 μL recombinant PLIN1 protein (Cat: TP306292, Origene) at 2.0 μg/ mL in 1X PBS (Cat: 524650, EMD Millipore,) was incubated overnight at 4 °C in a 96-well ELISA plate (Nunc MaxiSorpTM Flat-bottom, Cat: 439454, Thermo Fisher Scientific).

Techniques: Dominant Negative Mutation

Journal: Autoimmunity

Article Title: Anti-perilipin-1 autoantibodies in autoimmune Addison's disease and related endocrine disorders.

doi: 10.1080/08916934.2025.2461703

Figure Lengend Snippet: Figure 2. A longitudinal study of PLIN1-autoAbs for an APS-1 patient (patient #27, Table 2). PLIN1 indices of five serial samples from the same patient at five different time points in the period 2009–2021, and 21OH autoAbs indices plotted at the same time spanning. The thresholds for positivity to PLIN1-autoAbs and 21OH autoAbs are shown with horizontal dashed red and black lines, being 122, and 100 respectively.

Article Snippet: For coating of wells, 50 μL recombinant PLIN1 protein (Cat: TP306292, Origene) at 2.0 μg/ mL in 1X PBS (Cat: 524650, EMD Millipore,) was incubated overnight at 4 °C in a 96-well ELISA plate (Nunc MaxiSorpTM Flat-bottom, Cat: 439454, Thermo Fisher Scientific).

Techniques:

Journal: Autoimmunity

Article Title: Anti-perilipin-1 autoantibodies in autoimmune Addison's disease and related endocrine disorders.

doi: 10.1080/08916934.2025.2461703

Figure Lengend Snippet: Figure 3. Analysis of anti-PLIN1 IgM autoantibodies using ELISA. Sera samples of anti-PLIN1-autoAbs positive patients in radioimmunoassay were used to detect frequencies of IgM PLIN1-autoAbs through ELISA. (A) Frequency of anti-PLIN1 IgM autoAbs compared to control. (B) Serial sera samples from one patient with APS-1 (patient #27) analyzed for PLIN1- IgM autoAbs in ELISA. A commercial monoclonal antibody against PLIN1 was used as positive control. In (A) the blue dot depicts one blood donor scoring PLIN1 autoAbs borderline positive in RIA. In (A and B) the violet dot corresponds to the identical sample that has also been plotted in the PLIN+ in both the RIA and longitudinal groups.

Article Snippet: For coating of wells, 50 μL recombinant PLIN1 protein (Cat: TP306292, Origene) at 2.0 μg/ mL in 1X PBS (Cat: 524650, EMD Millipore,) was incubated overnight at 4 °C in a 96-well ELISA plate (Nunc MaxiSorpTM Flat-bottom, Cat: 439454, Thermo Fisher Scientific).

Techniques: Enzyme-linked Immunosorbent Assay, RIA Assay, Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Cyclophilin A in Arrhythmogenic Cardiomyopathy Cardiac Remodeling

doi: 10.3390/ijms20102403

Figure Lengend Snippet: Immunofluorescence staining of ACM RV section ( a ). Particular of a preadipocyte, positive for CD29 and perilipin 1 (PLIN1), expressing cyclophilin A (CyPA) ( b ). The image of merged signals is also shown.

Article Snippet: After washing in PBS, slides were blocked for 30 min in 10% goat serum-PBS (Sigma Aldrich, St. Louis, MO, USA) and incubated with specific primary antibodies against CyPA (1:100, sc-133494; Santa Cruz Biotechnology, Santa Cruz, CA, USA), PLIN1 (1:100, BP5015; OriGene, Herford, Germany) and CD29 (1:200, NCL-CD29; Leica, Wetzlar, Germany) in 2% goat serum-PBS O/N at 4 °C.

Techniques: Immunofluorescence, Staining, Expressing