Antibody to actin

Cells were lysed by using a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) sample loading buffer. The lysates were subjected to PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% nonfat dried milk and probed with the following specific primary antibodies: C/EBP‐β, sterol regulatory element binding protein (SREBP)‐1 (2A4), and fatty acid synthase (FASN) (A‐5; Santa Cruz); ATG5 (Proteintech, IL); and mammalian target of rapamycin (mTOR; 7C10) and phosphorylated‐mTOR (p‐mTOR; D9C2) (Cell Signaling Technology, Danvers, MA). After washing, the blots were incubated with secondary antibody for 1 hour. Proteins were detected by using an enhanced chemiluminescence western blot substrate (Pierce; ThermoFisher Scientific). Membranes were reprobed with antibody to actin (Santa Cruz) as an internal control for normalization of the protein load. ImageJ software (National Institutes of Health [NIH]) was used for densitometric scanning of western blot images.

For analysis of poly ADP-ribose polymerase (PARP) cleavage, light chain 3 (LC3), and glucose-regulated protein (Grp) 78 and Grp94 levels, proteins were separated on 8% denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinyl difluoride membrane, incubated with a 1:1,000 dilution of PARP C-2–10 monoclonal antibody or KDEL antibody to detect Grp78 and Grp94 from Enzo Life Sciences (Farmingdale, NY), LC3 antibody from Cell Signaling Technologies (Danvers, MA), and the antibody to actin used for normalization was from Santa Cruz Biotechnology (Dallas, TX). These antibodies were detected with a 1:10,000 dilution of the corresponding horseradish peroxidase-conjugated secondary antibody from Jackson ImmunoResearch Laboratories (West Grove, PA), and Enhanced Chemiluminescence reagents (EMD Millipore, Billerica, MA) and band intensities were recorded using a CCD camera-based imager (MicroChemi 4.2; FroggaBio, Toronto, ON, Canada). Where required, band intensities were corrected for unequal loading by reprobing the blots for actin.