Anti phospho gcn2

24S-OHC [52 (link)] was dissolved in EtOH (Wako, Osaka, Japan). F12511 was the generous gift of Kowa (Aichi, Japan). Thapsigargin and CHX were purchased from Wako (Osaka, Japan). GSK2606414 and ISRIB were from Cayman Chemical (Ann Arbor, MI, USA). GCN2iB was from MedChemExpress (Monmouth Junction, NJ, USA). Thapsigargin, GSK2606414, CHX, and ISRIB were dissolved in dimethyl sulfoxide (DMSO; Wako). The following antibodies were from commercial sources: anti-PERK (Cat# 3192), anti-phospho-eIF2α (Cat# 3398), anti-eIF2α (Cat# 5324), and anti-GCN2 (Cat# 3302) were from Cell Signaling (Danvers, MA, USA); anti-β-actin (Cat# A5441) was from Sigma-Aldrich (St. Louis, MO, USA); anti-TIA1 (Cat# 12133-2-AP), anti-ATF4 (Cat# 10835-1-AP), and anti-TDP-43 (Cat# 12782-2-AP) were all from Proteintech (Chicago, IL, USA); anti-G3BP1 (Cat# 611126) was from BD Biosciences (Franklin Lakes, NJ, USA); anti-phospho-GCN2 (Cat# ab75836) was from Abcam (Cambridge, UK); and anti-puromycin (Cat# MABE343) was from Merck Millipore (Burlington, MA, USA); All other chemicals, of analytical grade, were obtained from Sigma-Aldrich or Wako.

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).