Anti pparγ antibody e 8 sc 7273

Proteins from eWAT were extracted by macerating the tissue with a Polytron (PT1200 E) in a solution composed of 0.5 M EDTA 0.02% (v/v) pH8, 0.1% (v/v) 1 M Tris pH7.5, 0.0045% (m/v) sodium fluoride, 0.0019% (m/v) sodium orthovanadate, 10% Triton X-100, 0.02% (v/v) PMSF (serine protease inhibitor), 0.04% (v/v) Cip 25x (protein inhibitor cocktail), and water q.s.p., incubating it for 1 h on ice. Then, samples were centrifuged, and supernatant was collected for protein quantification using Pierce 660 nm Protein Assay Reagent (Thermo Scientific ). Electrophoresis gel was performed with 15 µg of protein and the following primary and secondary antibodies were used: anti-PPARγ (dilution 1:1000, Anti-PPARγ Antibody (E-8): sc-7273-Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-S273 PPARγ (dilution 1:200, BS-4888R, Bioss, Boston, MA, USA), anti-vinculin (dilution 1:1000, ab18058-Abcam, Waltham, MA, USA), anti-rabbit IgG (dilution 1:5000, A0545, Sigma-Aldrich, St. Louis, MO, USA), and anti-mouse IgG (dilution 1:5000, 401253, Calbiochem, San Diego, CA, USA). The final result was obtained using peroxidase reaction through Amersham ECL Prime Western Blotting Detection Reagent-GE in the ImageQuant LAS 500 (GE Health Care Life Sciences, Chicago, IL, USA) equipment, as described [14 (link)].