And gapdh

ATDC5 cells were incubated in L-DMEM with/without 1 μg/mL AdipoRon and increasing concentrations of d-glucose (5.5, 100, 150, and 200 mM) for 24 h. Cells were then lysed in icy RIPA buffer (Beyotime) for half an hour. Cell lysates were centrifuged at 12,000 rpm at 4 °C for 10 min, and the supernatants were stored at −20 °C for analysis. Equal amount of proteins (35 μg) were electrophoresed by 12% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% bovine serum albumin (BSA) for 2 h at room temperature, the membranes were incubated with anti-p-extracellular signal-related kinases (ERK) −1/2, anti-ERK1/2, and GAPDH (1:1,000, rabbit polyclonal antibodies, Cell Signaling Technology, Boston, MA, USA) for immunoblotting at 4 °C overnight. After rinsing three times and secondary antibodies for 1 h, immunoreactive bands were detected using a chemiluminescence gel imaging system (LAS4000 M; GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The ratio of the p-ERK to ERK was quantified using ImageJ v.1.4 analysis software (Bethesda, MD, USA).