Anti hri

Whole cells were lysed with M-PER buffer (Thermo Fisher Scientific) with a protease inhibitor cocktail (Roche Applied Science, Schlieren, Switzerland). Proteins were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to a nitrocellulose membrane (0.45 μm, Merck Millipore, MA, USA). The membrane was blocked with 5% skimmed milk (Rockland Immunochemicals, PA, USA) in PBST for 1 h, and then incubated with the primary antibodies overnight at 4℃. After washing, the blots were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, MA, USA) for 1 h at 20°C. The proteins were detected using the SuperSignal system (Thermo Fisher Scientific) with a chemiluminescence imaging system (Luminograph I, ATTO, Tokyo, Japan). The primary antibodies used in this study were anti-Tubulin (1:5000, Meridian Life Science, TN, USA), anti-eIF2α (1:500, Santa Cruz), anti-phospo-eIF2α (1:1000, Cell Signaling Technology), anti-PERK (1:500, Santa Cruz), anti-phospo-PERK (1:1000, Affinity Biosciences, OH, USA), anti-HRI (1:500, Santa Cruz), anti-γH2AX (1:1000, Abcam, Cambridge, Cambridgeshire, UK). Immunoblot bands were quantified using NIH ImageJ software (Bethesda, MD, USA).

Phospho-specific anti-eIF2α and the pan anti-eIF2 were purchased from Cell Signaling Technology (Beverly, MA). Anti-FMRP, anti-FXR1, anti-eIF4E, anti-eIF4GI and anti-G3BP1 antibodies have been previously described [3 (link)]. Anti-RCK, anti-ATF4, and anti-HRI antibodies were purchased from Santa Cruz Biotech. Anti-PERK and anti-tubulin antibodies were obtained from Abcam. Anti-puromycin antibodies were obtained from EMD Millipore.