Immunoblotting (in vivo samples)

Protein (8 ug) was loaded on 4–15% Criterion Tris/Glycine gels (Bio-Rad, #5671085) and run for 60 min at 150 constant volts in 1X Tris/Glycine buffer (Bio-Rad, #1610732). Protein was transferred from gels to nitrocellulose with Bio-Rad Turbo on default setting. All blots were air-dried, rehydrated with TBS and blocked for 1 h at RT in 5% BSA in TBST (0.1%). Blots were exposed to primary antibody in 5% BSA in TBST (0.1%) overnight at 4 C. Blots were washed 3× with TBST (0.1%), 5 min per wash, at RT. Secondary antibody was added at 1:18,000: anti-rabbit-HRP (CST 7074) and/or anti-mouse-HRP (CST 7076) in 5% BSA in TBST (0.1%). Blots were incubated at RT for 1 h. Blots were washed 3 times in TBST (0.1%) for 5 min each wash at RT. All incubations and washing were done while rocking. Signal was developed with 6 ml of Femto Max ECL substrate (ThermoFisher, #34094) for 4 min and blots read on ChemiDoc. Densitometry was performed with ImageLab.