Anti p150

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).