Total egfr and total erk antibodies

Proteins were extracted from cells and tissues using cell lysis buffer (Cell Signaling) supplemented with proteinase (Roche) and phosSTOP phosphatase inhibitor cocktail (Roche). Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which was then probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody, and visualized by ECL (Pierce). Antibodies specific for p-MEK 1/2, total MEK 1/2, p-ERK, p-AKT (Ser473), total AKT, PTPN9, and beta-actin were obtained from Cell Signaling Technologies. Total EGFR and total ERK antibodies were purchased from Santa Cruz Biotechnology. Antibody specific for p-EGFR (1173) was obtained from Novus Biologicals, and antibodies specific for Beta-Tubulin was from Milipore.

Treated 2341 cells were stimulated with EGF (20ng/ml) for 20 min prior to protein extraction which was performed using cell lysis buffer (Cell Signaling) supplemented with proteinase and phosSTOP phosphatase inhibitor cocktail (Roche). Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were then probed with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibody, and visualized by ECL (GE Healthcare). Antibodies specific for p-EGFR (Tyr1068), p-Akt (Ser473), total Akt, p-ERK, and beta-actin were from Cell Signaling Technology. Total EGFR and total ERK antibodies were from Santa Cruz Biotechnology, Inc.

Proteins were extracted from cells using cell lysis buffer (Cell Signaling Technology, Danvers, Massachusetts, USA) supplemented with proteinase (Roche, Upper Bavaria, Germany) and phosSTOP phosphatase inhibitor cocktail (Roche). Proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were then probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody, and visualized by ECL (GE Healthcare, Buckinghamshire, UK). Antibodies against phospho-ERK, phospho--Akt, total Akt, phosphor-CRAF, total CRAF, phospho-IGF1R, total IGF1R, phospho-MEK, total MEK, phospho EGFR, Ras, phospho Axl, total B-catenin were purchased from Cell Signaling Technology. Total EGFR and total ERK antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and total Axl was obtained from R&D Systems (Minneapolis, Minnesota, USA). Antibody specific for p-EGFR (1173) was obtained from Novus Biologicals, and antibodies specific for Beta-Tubulin was from Milipore. Anti-Wnt5A antibody was purchased from Abcam (Cambridge, Massachusetts, USA).