Pan eif2α

MDA-MB-231 or NHDF cells were treated with the indicated concentrations of CM16 for 60 min and lysates were collected by washing the cells with ice cold PBS followed lysis using a buffer composed of 20 mM HEPES [pH 7.5], 150 mM NaCl, 1% Triton-X100, 10% glycerol, 1 mM EDTA, 10 mM tetrasodium pyrophosphate, 100 mM NaF, 17.5 mM β-glycerophosphate, 1 mM PMSF, 4 mg/ml aprotinin, and 2 mg/ml pepstatin A. Samples were resolved on a 10% NuPAGE gel and transferred to a PVDF membrane (BioRad) for immunoblotting. The following antibodies were used in this study: eukaryotic elongation factor 2 (eEF2) (#9742, Cell Signaling), pan eIF2α (#9722, Cell Signaling), phospho-eIF2α (Ser51) (#9721, Cell Signaling). Two independent experiments were performed.

Cells were lysed in RIPA buffer (20 mM Tris [pH 7.6], 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % NP40, 0.5 % sodium deoxycholate, 0.1 % SDS, 10 mM NaF, 20 mM β-glycerophosphate, 1 mM PMSF, 4 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin), resolved on a 10 % SDSAPAGE, and transferred to PVDF membranes (Bio-Rad). Antibodies used in this study were directed to: MYC (Santa Cruz, SC-40), GFP (Cell Signaling Technology, 2555), eEF2 (Cell Signaling Technology, 2332), NFAT (BD Biosciences, 610702), H3K27ac (Abcam, 2118291), Histone H3 (Abcam, 8898), GAPDH (Abcam, 8245), p-ERK 1/2 (Cell Signaling Technology, 9101), pan-ERK 1/2 (Cell Signaling Technology, 9102), p-eIF2 (Cell Signaling Technology, 3597) and pan eIF2α (Cell Signaling Technology, 9722).