Westernbright ecl western blotting hrp substrate

Total protein was prepared using RIPA (Thermo Fisher Scientific). The plasma membrane protein was prepared using a membrane protein extraction kit (Thermo Fisher Scientific) and the tissue nuclear protein was prepared using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). The protein was separated by electrophoresis on SDS-polyacrylamide gels and immunoblotted with antibodies against pAkt^ser473 (Cell Signaling Technology, MA, USA, #4060, 1:500), Akt (CST, #9272, 1:2000), Glut4 (sc-53566, 1:500), Insr (CST, #3025, 1:1000), pInsr^Tyr1150/1151 (CST, #3024, 1:500), Irs1 (sc-515017, 1:500), pIrs1^Tyr608 (Merck Millipore, 09-432, 1:1000), Txnip (CST, #14715, 1:1000), Keap1 (CST, #8047, 1:1000), Nrf2 (Abcam, ab137550, 1:2000), Dnmt3a (CST, #3598, 1:1000) , Lamin B1 (CST, #13435, 1:1000) and Gapdh (CST, #2118, 1:2000). The antibody-reactive bands were visualized using ECL chemiluminescence detection system (WesternBright™ ECL Western Blotting HRP Substrate, Advansta, CA,USA) and the band intensity was quantified by Image J software (National Institutes of Health).