Chop ab11419

Cells were washed with phosphate-buffered saline (PBS) and harvested using a lysis buffer containing 50 mM Tris-HCl pH 8.0, 1% SDS, and previously described protease and phosphatase inhibitors [25 (link)]. Equivalent amounts of protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes (EMD Millipore; Billerica, MA). The membranes were incubated with antibodies against total and phosphorylated eukaryotic initiation factor-2α (eIF2α sc-133132; Santa Cruz Biotechnology, Santa Cruz, CA; p-eIF2α NB110-56949; Novus Biologicals, Littleton, CO), or C/EBP homologous protein (CHOP #ab11419; Abcam, Cambridge, UK). The following day, membranes were washed in Tris-buffered saline and Tween 20 (TBST) pH 7.4, incubated for 1 hour with secondary detection antibodies ((IRDye 680 CS-conjugated anti-rabbit, IRDye 800 CS-conjugated anti-rabbit, IRDye 680 CS-conjugated anti-mouse, or IRDye 800 CW-conjugated anti-mouse (LI-COR Biosciences, Lincoln, NE)), washed in TBST, and scanned for infrared signal using the Odyssey Imaging System (LI-COR Biosciences). Density was quantified using Image Studio (LI-COR Biosciences).