Anti puromycin

Thapsigargin (Tg), puromycin dihydrochloride, p38 inhibitor (SB203580), and JNK inhibitor (SP600125) were purchased from Sigma (St. Louis, MO, USA). AZD8055 and refametinib were procured from Cayman Chemical (Ann Arbor, MI, USA). The anti-puromycin and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Millipore (Burlington, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. The anti-phospho 4E-BP1 (Ser65), anti-4E-BP1, anti-phospho ERK 1/2, anti-ERK 1/2, anti-phospho p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho eIF2α, anti-eIF2α, anti-phospho c-Jun, anti-c-Jun, and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor^® 488 AffiniPure Goat Anti-Mouse IgG (Fcγ fragment specific) was supplied by Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The Selleck Anti-Cancer Compound Library consisting of 414 drugs was purchased from the Department of Convergence Medicine, ASAN Medical Center, University of Ulsan College of Medicine (Seoul, Korea).

Anti‐HA: Invitrogen (Cat. No. 26183, 1:10,000 dilution), anti‐GFP: Invitrogen, (Cat. No. A‐6455, 1:1,000 dilution), anti‐RFP: Invitrogen, (Cat. No. MA5‐15257, 1:1,000 dilution), anti‐PABP: Sigma‐Aldrich, (Cat. No. P6246, 1:2,000 dilution), anti‐puromycin: (Millipore, [Cat. No. MABE341, 1:100,000 dilution], anti‐vinculin: Sigma‐Aldrich, [Cat. No. V9264, 1:1,000 dilution], anti‐β‐actin: Santa Cruz, [Cat. No. sc‐47778, 1:500 dilution], anti‐myc: Roche [9E10], 1:300 dilution).

OPCs were washed twice with PBS and lysed in ice-cold RIPA buffer (Sigma) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, PI78441). Lysates were then centrifuged at 12,000 rpm for 20 min at 4°C. A total 20 µg of protein lysates was separated by 4–12% SDS-PAGE (Bio-Rad, 4561095) and transferred to a nitrocellulose membrane. The following primary antibodies were used: anti-p-eIF2α (Abcam, ab32157, 1:2000), anti-T-eIF2α (Cell Signaling, 9722s, 1:1000), anti-puromycin (Millipore, MABE343, 1:2000), anti-BIP (Cell Signaling, 3177s, 1:1000), anti-GADD34 (Proteintech, 10449–1-AP, 1:500), anti-ATF4 (Santa Cruz, sc-390063, 1:500), anti-CHOP (Thermo Fisher, MAI-250, 1:500), anti-XBP-1-spliced (Cell Signaling, 82914s, 1:1000), and anti-actin (Sigma, A2066, 1:2000). Quantification of Western blot bands were performed by Image Lab Software (Bio-Rad).

For whole cell extracts, cells were lysed in EBC buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with protease and phosphatase inhibitors. Samples were mixed with Laemmli sample buffer and boiled for 5 min. Protein samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in PBS-Tween + 5% milk before incubation with primary antibody overnight at 4 °C. Next, membranes were washed in PBS-tween and incubated with HRP Goat Anti-Rabbit or Goat Anti-Mouse IgG Antibody (H + L) for 1 h at room temperature. Lastly, membranes were washed in PBS-tween and visualized by chemiluminescence (Clarity Western ECL substrate, Bio-Rad) using the Bio-Rad Chemidoc imaging system. Antibodies used in this study were: anti-phospho-p38 (Cell Signaling, #9216), anti-p38 (Cell Signaling, #9212), anti-phospho-SAPK/JNK (Cell Signaling Technology, #9255), anti-ZAK (Proteintech, #14945-1-AP), anti-ZAKα (Bethyl #A301-993A), anti-p150 (BD biosciences, #610473), anti-phospho-GCN2 (Abcam, #ab75837), anti-phospho-eIF2α (Cell Signaling, #3398), anti-puromycin (Millipore, #MABE343), anti-EDF1 (Abcam, #ab174651), anti-RPS2 (Bethyl, #A303-794A), anti-RPS10 (Abcam, #ab151550), anti-ZNF598 (Abcam, #ab111698), ASK1 (Thermo Scientific, #702278), α-tubulin (Sigma, #T9026) and anti-HA (Santa Cruz, #sc-7392).