Adiponectin Receptor and AMPK Signaling

Cell homogenates (20 μg/well) were loaded onto 10% SDS polyacrylamide gels in denaturing conditions at 80 mA for 90 min and transferred electrophoretically (100 mA/blot, 2 h; Power Pack; Bio-Rad Laboratories, Inc., USA) to polyvinylidene fluoride (PVDF) membrane. Immunoblotting was performed as described previously [48 (link)]. Nonspecific binding was blocked with 5% non-fat milk powder in Tris-buffered saline-Tween containing 0.1% Tween-20 (PBS-T) for 1 h. Primary antibodies including rabbit anti-AdipoR1 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-AdipoR2 (1:1000, Boster Biological Technology, USA), rabbit anti-AMPK (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-p-AMPK^T172 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-α-Tubluin (1:5000, Cell Signaling Tech. Inc., USA), rabbit anti-p-NF-κB p65^S536 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-NF-κB p65 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-p-IκBα (Ser32) (1:1000, Cell Signaling Tech. Inc., USA), mouse anti-IκBα (1:1000, Cell Signaling Tech. Inc., USA) antibody were incubated at 4 °C overnight, followed by HRP-conjugated secondary antibodies (goat anti-rabbit, 1:5000 or rabbit anti-mouse, 1:5000; Dako, Glostrup, Denmark) at RT for 1 h. The immunoblot signals were visualized by Westernbright Quantum HRP substrate (advansta, USA).