Anti egfr

Total cellular proteins were extracted by solubilizing the cells in EB buffer (50 mmol/L Hepes pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 5 mmol/L EDTA, 2 mmol/L EGTA; all reagents were from Sigma-Aldrich (St. Louis, MO, USA), in the presence of 1 mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride, and a mixture of protease inhibitors. Extracts were clarified by centrifugation and normalized with the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). Western blot detection was performed with enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA) and peroxidase conjugated secondary antibodies (Amersham, Little Chalfon, UK). The following primary antibodies were used for Western blotting (all from Cell Signaling Technology, Danvers, MA, USA, except where indicated): anti-phospho-p44/42 ERK (Thr202/Tyr204; 1:1000); anti-p44/42 ERK (1:1000); anti-phospho EGFR (Tyr1068) (1: 1000); anti-EGFR (EnzoLifeSciences, Farmingdale, NY, USA) (1:1000); anti-ferritin light chain (Abcam, Cambridge, UK) (1:1000); anti-Actin (Santa Cruz Biotechnology, Dallas, TX, USA) (1:1000).

The sources of antibodies and reagents are as followed: anti-pY291 Fas (prepared as previously described¹³ and validated for immunoblotting¹³ and flow cytometry Fig. S8); anti-Src, anti-Yes (R&D Systems); anti-human Fas (C20, for immunoblotting) and anti-Cyclin D1 (Santa Cruz); anti-Fas (Apo1–3, for co-immunoprecipitation) (Enzo); anti-EGFR, anti-pY1068 EGFR, anti-pY705 STAT3, anti-STAT3, anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pErk1/2, and anti-Histone H3 (Cell Signaling Technology); anti-Erk1/2 (Sigma); anti-GAPDH (Calbiochem); horseradish peroxidase conjugated anti-rabbit and anti-mouse (Jackson ImmunoResearch); anti-mouse IgG-Alexa Flour 488 (Life Technology); Recombinant human EGF (Gibco), recombinant amphiregulin (R&D Systems); protein G sepharose beads (Zymed Laboratories); WST-1 (Interchim); Matrigel (BD Bioscience). Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection according to the manufacturer’s protocol for reverse transfection.