Anti egfr clone13g8

Total cellular proteins were extracted by solubilizing the cells in cold EB buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA and 2 mM EGTA; all reagents were from Sigma-Aldrich, except for Triton X-100 from Fluka) in the presence of 1 mM sodium orthovanadate, 100 mM sodium fluoride and a mixture of protease inhibitors (pepstatin, leupeptin, aprotinin, soybean trypsin inhibitor and phenylmethylsulfonyl fluoride). Extracts were clarified by centrifugation, and protein concentration was determined using BCA protein assay reagent kit (Thermo). Western blot detection was performed with enhanced chemiluminescence system (GE Healthcare) and peroxidase-conjugated secondary antibodies (Amersham). Chemoluminescent signal was acquired by the LAS4000 Image Reader (Fujifilm). The following primary antibodies were used for western blotting (all from Cell Signaling Technology, except where indicated): anti-phospho p44/42 ERK (Thr202/Tyr204); anti-p44/42 ERK; anti-phospho-AKT (Ser473); anti-AKT; anti-phospho EGFR (Tyr1068, Abcam); anti-EGFR (clone13G8, Enzo Life Sciences); and anti-vinculin (Sigma-Aldrich). All the antibodies were diluted 1:1,000 except for total EGFR (1:100) and vinculin (1:2,000). Full-length blot can be viewed in Supplementary Fig. 6.