Eif2ak3

TC-71 and MHH-ES-1 cells were incubated with viscum, TT or viscumTT in increasing concentrations for 24 h. The cells were washed twice with phosphate-buffered saline and incubated in Lysis Buffer 17 (R&D systems, Minneapolis, MN) containing protease inhibitors (complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics GmbH) to obtain cell lysates. Protein concentration was determined using Bradford solution (Bio-Rad, Munich, Germany). TC-71 and MHH-ES-1 cell lysates (30 μg protein/lane) were separated on SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad system) and blocked with 5% non-fat milk in 50 mM Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Blots were incubated overnight at 4 °C in TBST containing 5% BSA and primary antibody, washed thrice in TBST and incubated 1 h with HRP-conjugated secondary antibodies (anti-rabbit and anti-mouse, Bio-Rad) then visualized by ECL (Thermo Scientific) on a Molecular Imager ChemiDoc (Bio-Rad). Primary antibodies were directed against p-MAPK14 (Thr180/Tyr182, #9211 Cell Signaling Technology, Danvers, MA, USA), LC3B (#2775 Cell Signaling Technology), EIF2AK3 (#3192, Cell Signaling Technology), p-MAPK8 (sc-6254, Santa Cruz biotechnology, Dallas, TX, USA), HSPA5 (#G8918, Sigma-Aldrich) and ß-actin conjugated directly to peroxidase (#A3854, Sigma-Aldrich).