Egfr 06 847

Liver tissues and cells were homogenized in Nonidet P40 lysis buffer (1% NP40, 50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.05% SDS, 0.5 mM Na-vanadate, 100 mM NaF, 50 mM β-glycerophosphate, 1 mM PMSF) that was supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, USA). The homogenates were centrifuged at 12,000g for 15 min at 4°C, and the supernatants were collected. The liver lysates or cell lysates were used for Western blotting, as described (Choi et al., 2017 (link)). The following were purchased from Sigma-Aldrich (USA): α-Tubulin (T5168), β-Actin (A5441), Flag (F1804). The following were purchased from Santa Cruz Biotechnology (USA): eIF2α (SC-133132), STAT3 (SC-482), STAT5 (SC-835). The following were purchased from Cell Signaling Technology (USA): Phospho-EGFR (Y1068) (#3777), AKT (#4691), Phospho-AKT (S473) (#4060), ERK (#4695), Phospho-ERK (T202/Y204) (#4407), Phospho-STAT3 (Y705) (#4113), Phospho-STAT5 (Y694) (#9314). The following were purchased from Merck Milipore (USA): EGFR (06-847). The following were purchased from Abcam (UK): p-eIF2α (S31) (ab32157). The secondary peroxidase-conjugated antibodies were purchased from Thermo Fisher Scientific or Jackson ImmunoResearch (USA).