Protein Immunoblot Analysis of Mouse Liver

Membrane and nuclear proteins were prepared individually from frozen livers, and equal amounts of protein from each mouse of the same group were pooled as previously described (Engelking et al., 2004 (link)). Aliquots of pooled proteins were subjected to SDS-PAGE on 8% gels and transferred to a nitrocellulose membrane (Bio-Rad, USA). Immunoblot analyses were performed using polyclonal anti-mouse SREBP-1, SREBP-2, and HMG-CoA reductase antibodies as previously described (Rong et al., 2017 (link)). Anti-Scavenger receptor class B type 1 (SR-BI) antibody was obtained from Abcam (USA) and anti-cluster of differentiation 36 (CD36) antibody was obtained from R&D Systems (USA). Anti-mouse cAMP response element binding protein (CREB; Invitrogen, USA) and anti-dog calnexin (Enzo Life Science, USA) antibodies were used as loading controls for nuclear and membrane proteins, respectively. Signals were detected using the SuperSignal West Pico Chemiluminescent Substrate System (Thermo Fisher Scientific) and visualized with Odyssey CLx Imaging system and LI-COR Image Studio^TM software (LI-COR, USA).