Proteins were extracted from the BMSCs from each group using RIPA buffer, according to the manufacturer’s instructions. A BCA Protein Assay kit (Beyotime, Beijing, China) was used to determine the protein concentrations. Equal amounts of proteins were loaded onto sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the primary antibodies overnight at 4 °C (1:300 dilution, monoclonal mouse anti-PPARγ, anti-GREM1, anti-Runx2, anti-Bmp-2, or anti-C/EBPα; Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with a secondary antibody (1:3,000 dilution, horseradish peroxidase-conjugated rabbit anti-mouse IgG, Santa Cruz Biotechnology, Santa Cruz, USA) at 37 °C for 1 h. The blots were examined using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). An antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, USA) served as an internal reference.
Mouse monoclonal anti pparγ
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse monoclonal anti-PPARγ is a laboratory reagent used to detect and study the peroxisome proliferator-activated receptor gamma (PPARγ) protein in various experimental models. This antibody is produced using mouse hybridoma technology and specifically binds to the PPARγ protein, allowing for its identification and quantification in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence detection kit, by Cytiva (2 mentions)
Anti c ebpα, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Horseradish peroxidase conjugated mouse anti rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Antibody against gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti runx2, by Santa Cruz Biotechnology (1 mentions)
Anti bmp2, by Santa Cruz Biotechnology (1 mentions)
Antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti nfatc1, by Santa Cruz Biotechnology (1 mentions)
Rabbit polyclonal anti pparγ, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti lamin a c, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Monoclonal mouse anti β actin, by Proteintech (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Tissue rna kit, by Omega Bio-Tek (1 mentions)
5 protocols using mouse monoclonal anti pparγ
1
Quantification of Osteogenic Markers
2
Western Blot Analysis of Osteogenic Markers
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Total proteins of cultured cells were extracted using RIPA buffer containing phenylmethanesulfonylfluoride. Protein concentration was determined using the BCA protein assay kit (Beyotime, Haimen, China) according to the manufacturer’s protocol. Proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were incubated overnight at 4°C with diluted (1:300) primary antibodies (monoclonal mouse anti-PPARγ or anti-C/EBPα or anti-OCN or anti-Runx2; Santa Cruz Biotechnology, Dallas, TEX, USA). Following extensive washing, the membranes were incubated with diluted (1:3000) horseradish peroxidase-conjugated rabbit anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TEX, USA). Signals were determined using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). An antibody against β-actin (Santa Cruz Biotechnology, Dallas, TEX, USA) served as endogenous reference.
3
Western Blot Analysis of NFAT, PPAR, and Calcineurin
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Whole cell lysates or immunoprecipitated complexes were prepared in RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL, USA) with 1x protease inhibitor cocktail (Sigma‐Aldrich, St. Louis, MO, USA). Nuclear and cytoplasmic extracts were prepared using a commercial kit (G‐Bioscience, St. Louis, MO, USA). Immunoblots were performed by routine procedures as described previously 24 . Blots were probed with mouse monoclonal anti‐NFATc1(1 : 1000, Santa Cruz, Dallas, TX, USA) and rabbit polyclonal anti‐NFATc1 (1 : 1000, Santa Cruz), mouse monoclonal anti‐PPARγ (1 : 1000, Santa Cruz) and rabbit polyclonal anti‐PPARγ (1 : 1000, Santa Cruz), rabbit polyclonal anti‐calcineurin Aα (catalytic subunit A isoform alpha; 1 : 3000, Santa Cruz) and anti‐calcineurin B2 (regulatory subunit B type 2; 1 : 3000, Flarebio, College Park, MD, USA), mouse monoclonal anti‐lamin A/C (1 : 3000, Cell Signaling, Danvers, MA, USA), or rabbit polyclonal anti‐β‐actin (1 : 5000, Cell Signaling).
4
Evaluating PPARγ Protein Levels in Adipose Tissue
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The protein level of PPARγ in inguinal WAT (iWAT) was assessed by western blot using mouse monoclonal anti- PPARγ (Santa Cruz, Cat. No. sc-7273, 1:1000) (Chakraborty et al., 2019 (link); Jung et al., 2019 (link)) and mouse monoclonal anti-β-actin (Protein Tech, Cat. No. 60008-1-Ig, 1:2000). Total RNA was isolated from liver and fat tissues using Tissue RNA kit (Omega Bio-Tek, GA). The first strand cDNA was reverse-transcribed using TAKARA reverse transcription kit. Real-time quantitative PCR reactions were performed with SYBR Premix Ex TaqTM (TAKARA) on a CFX96™ Real-Time PCR Detection System (Bio-Rad). Relative mRNA expression levels were normalized to β-actin levels. The sequences of the primers used were listed in Supplementary Table 1
5
Corneal Expression of NOX and PPAR Isoforms
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The corneal expression of NOX and PPAR isoforms was evaluated by immunohistofluorescence in deparaffinized eye sections. Diva Decloaker (Biocare Medical, LLC, Pacheco, CA), an antigen retrieval reagent, and the following specific primary antibodies were used for immunostaining: mouse monoclonal anti-NOX1 (Santa Cruz Biotechnology, Santa Cruz, CA; Cat. No. sc-518023; 1:200 dilution); rabbit monoclonal anti-NOX2 (Epitomics-Abcam, Burlingame, CA; Cat. No. ab129068; 1:100 dilution); rabbit monoclonal anti-NOX4 (Epitomics-Abcam; Cat. No. ab133303; 1:500 dilution); mouse monoclonal anti- PPARα (Santa Cruz Biotechnology; Cat. No. sc-398394; 1:200 dilution); mouse monoclonal anti-PPARγ (Santa Cruz Biotechnology; Cat. No. sc-271392; 1:200 dilution). Goat anti-rabbit Alexa Fluor® 555 (Cohesion Biosciences Ltd., London, UK; Cat. No. CSA3411) and Goat anti-mouse Alexa Fluor® 647 (Cat. No. CSA3808) were used as fluorescent secondary antibodies, where appropriate, and sections were mounted with DAPI Fluoromount-G®. Image J-NIH freeware (v. 2.0.0) was used to measure the intensity of the staining on parallel images of both animal groups, which were acquired from central corneal sections and processed with the same settings. Results were expressed as relative to the control group.
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