EGFR tyrosine phosphorylation assay

Subconfluent AGS cells cultured in RPMI-1640 medium were switched to serum-free medium for 8 h and then treated with CM or fresh medium for 45 min. Anti-IL-8 antibody (to neutralize IL-8) was added to the cells for 30 min before stimulation. Cells were lysed with lysis buffer (137 mM NaCl, 2.7 mM KCl, 10 mM PO₄³⁻, 1% Triton X-100 and 1 mM EDTA) containing protease and phosphatase inhibitor cocktails (Sigma) and then sonicated. The cell debris was removed by centrifugation, and the supernatant was used for immunoprecipitation assays using an anti-EGFR (EP38Y) antibody. Complexes of antibody and antigen were washed four times with lysis buffer. The immunoprecipitates were subjected to SDS-PAGE followed by immunoblot analysis using an anti-phospho-EGFR antibody.