Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Chemerin is resident to vascular tunicas and contributes to vascular tone

doi: 10.1152/ajpheart.00239.2023

Figure Lengend Snippet: A. Detection of chemerin protein expression in tunicas of the thoracic aorta from Dahl SS male and female rats. P = PVAT, A = adventitia; M = media, E = endothelium, L = lumen. The first row demonstrates colocalization of chemerin signal (green) with smooth muscle cell (SMC) a-actin (red) and second row with the adipocyte marker perilipin (red). DAPI (blue) is shown in the leftmost box, for each image. Images are representative of 4/5 different rats. B. Detection of chemerin protein in the human epigastric artery. Images are representative of 3 different human samples. Negative controls shown in bottom left corner for each image. 20x images, scale bar = 100 μm

Article Snippet: Sections were then species-specific blocked with protein blocker for one hour, followed by an incubation for 24 hours with chemerin (cat. #H-002–52, rabbit TIG-2; 1:200, Phoenix Pharmaceuticals Inc, Belmont, CA, USA), alpha actin (cat. # C6198, alpha actin-Cy3, incubated simultaneously with chemerin,1:1000, Sigma-Aldrich, St. Louis, MO, USA), or perilipin (cat. # AM09128SU-N, incubated sequentially prior to chemerin, ready to use, Origene, Herford, Germany) or no primary antibody at 4 °C in a humidified, closed chamber.

Techniques: Expressing, Marker

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

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

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: 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