Western Blot Analysis of Cellular Proteins

Equal amounts of protein lysates were separated by SDS-PAGE, and immunoblot analyses were carried out for three independent experiments using horseradish peroxidase–tagged secondary antibody. Primary antibodies used for immunoblots included total eIF2α (Cell Signaling Technology, #9722), eIF2α-P (Cell Signaling Technology, #9721), custom affinity-purified ATF4 antibody [26 (link)], CAT1 (Abcam, ab37588), GAPDH (Abcam, ab9485), puromycin (EDM Millipore, #17H1), and p70 S6 Kinase (49D7) rabbit mAb (Cell Signaling Technology #2708). Blots were incubated with Pierce ECL Western Blotting Substrate prior to imaging on FluorChem M- Multiplex fluorescence (Protein Simple).