Eif2α sc 133132

PFOA (96%), Thapsigargin (TG), Ionomycin (ION), Thiazolyl Blue Tetrazolium Bromide, Ceapin-A7, 4-Phenylbutyric acid (4-PB), N-acetyl-L-cysteine (NAC), 4,5-Dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate (Tiron), Hydrogen peroxide (30%), 2-aminoethoxydiphenyl borate (2-APB) and Dantrolene (Dan) were purchased from Sigma-Aldrich (St. Louis, MO). PERK Inhibitor I (GSK2606414), PERK Inhibitor II (GSK2656157), IRE1 Inhibitor III (4µ8c), and IRE1 Inhibitor IV (KIRA6) were purchased from Millipore Sigma (Burlington, MA). Tunicamycin (TM) was purchased from Santa Cruz Biotechnology (Dallas, TX) and Palmitoleic acid (POA) was purchased from Cayman Chemical (Ann Arbor, MI). Cell culture media and antibiotics were purchased from Sigma-Aldrich (St. Louis, MO) and fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT). The following primary antibodies were used for Western analysis: phospho-eIF2α (3597) and Chop (2895) from Cell Signaling Technology (Danvers, MA); eIF2α (sc-133132) and Gapdh (sc-365062) from Santa Cruz Biotechnology (Dallas, TX); and Atf4 (10835-1-AP) from Proteintech Group (Rosemont, IL). Secondary antibodies against mouse or rabbit species were conjugated to HRP and obtained from Cell Signaling Technology (Danvers, MA).

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).