Western Blot Analysis of Adipogenesis Regulators

Homogenized cells were ice-cold lysed by ice-cold RIPA buffer [50 mM Tris-Cl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.5% Sodium deoxycholate, 1% Triton X-100, 50 mM β-glycerophosphate, 50 mM NaF, and 1 mM Na₃VO₄] containing protease inhibitor (Roche, Switzerland). After centrifugation at 13,000 rpm for 15 min, supernatants were transferred to a new tube. Protein concentrations were measured using the Bradford assay (Bio-Rad, USA). Western blot analysis was performed using standard protocols (41 (link)). The following primary antibodies were used: PPARγ (2435s, CST), C/EBPα (2295, CST), C/EBPβ (sc7962, Santa Cruz), FABP4 (2120s, CST), Akt (9272s, CST), p-Akt (9271s, CST), AMPKα (2532s, CST), p-AMPKα (2535s, CST), β-Catenin (06-734, Sigma), p38 (9212s, CST), p-p38 (9215s, CST), ERK (9102s, CST), p-ERK (9106s, CST), JNK (9252s, CST), p-JNK (9251s, CST), CHOP (2895s, CST), and HSP90 (sc-13119, Santa Cruz). Specific antibody signals were detected by horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG antibody (Santa Cruz) and detected with an enhanced chemiluminescence system (Fusion Solo S, Vilber Lourmat, France). The relative amounts of each protein band were quantified with the ImageJ software.