Enhanced chemiluminescent autoradiography

Cells were lysed in radioimmunoprecipitation lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate) containing proteinase inhibitor cocktail (MedChemExpress, Monmouth Junction, NJ, USA). The protein samples were electrophoresed through 10% SDS polyacrylamide gels and transferred onto polyvinyl difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin at room temperature for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight, and then incubated with secondary antibodies at room temperature for 1 h. Immunoreactivity was detected with enhanced chemiluminescent autoradiography (Millipore). Chemiluminescence was determined using the AI600 System (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The antibody against GAPDH (60004-1-Ig, dilution 1:1000) was purchased from Proteintech. Antibodies against HSPA8 (8444, dilution 1:1000) and DEK (29812, dilution 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

WAT proteins were extracted using a lysis buffer containing protease inhibitors (ST505, Beyotime Biotechnology) and phosphatase inhibitors (P1082, Beyotime Biotechnology). Protein samples were resolved by SDS–PAGE (8%) and immunoblotted onto polyvinylidene difluoride membranes. Immunoblotting was conducted using extracellular signal-regulated protein kinase 1/2 (Erk1/2) (Cell Signalling Technology, 1:1,000 dilution, 9102) and phosphorylated Erk1/2 (Thr202/Tyr204) (Cell Signalling Technology, 1:1,000 dilution, 9101). Immunoreactivity was detected using enhanced chemiluminescent autoradiography (Millipore). Band quantification was performed using ImageJ software.