Bs 4888r

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)].

The damaged hippocampal region tissues were separated with RIPA buffer (Beyotime, China) on ice and homogenized to extract protein. Forty μg proteins were separated with 12% SDS-PAGE (Bio-Rad, China) and transferred to PVDF membranes (EMD Millipore). After blocking with 5% milk for 1.5 h, the membranes were incubated with appropriate primary antibodies diluted with 5% BSA for overnight at 4 °C. The primary antibodies consisted of phospho-PPARγ (ser273) (1:1000, bs-4888R, Bioss, China), LC3BI/II (1:1000, 4108, Cell signaling technology, China), phospho-NF-κB p65 (1:800, bs-230303R, Bioss, China), GAPDH (1:1000, bs-0755R, Bioss, China). After washing with TBS-0.01% Tween 20 for 3 times (10 min/wash), the secondary antibody Goat Anti-rabbit lgG/HRP (1:1000, bs-0295G-HRP, Bioss, China) was cultured with the membranes for 2 h at 25 °C. After washing, the signals were visualized using enhanced chemiluminescence reagent (D085075, Bio-Rad, China).