Phospho p44 42 mapk erk1 2 thr202 tyr204 rabbit mab

Western blot analysis of cultured HNSCC cells was performed to measure the expression and phosphorylation of EGFR and related signaling molecules. Western blotting was also performed to demonstrate whether erlotinib is able to inhibit the phosphorylation of EGFR. HNSCC cells were treated with 1 M erlotinib for 4 hours and stimulated with EGF (10 ng/mL; Upstate Biotechnology) for 15 minutes before harvesting protein. To compare molecular changes in the EGFR downstream signaling molecules, including AKT and MAPK, Western blot analysis was performed after 48 hours of treatment with 1 µM erlotinib. These downstream pathways were also examined after transient transfection of HRAS small interfering RNA (siRNA) into erlotinib-resistant HN31 and UM-SCC-19 cells. HRAS expression was measured using Western blotting after stable transfection of activated HRAS mutation constructs into erlotinib-sensitive HN5, UM-SCC-10B, and UM-SCC-22A cells and stable transfection of HRAS short hairpin RNA (shRNA) into HN31 cells.
The primary antibodies used in Western blotting consisted of an EGFR rabbit monoclonal antibody (mAb), an HRAS (C–20) rabbit polyclonal antibody (Santa Cruz Biotechnology), a phospho-EGFR (Tyr1068) rabbit mAb, an AKT rabbit mAb, a phospho-AKT (Ser473) rabbit mAb, a p44/42 MAPK (Erk1/2) mouse mAb, and a phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit mAb (Cell Signaling).