Anti human pegfr antibody

Lysates of cell lines and xenograft tissues were generated using lysis buffer as previously reported (25 (link)). The lysate was centrifuged at 16,000 g at 4°C for 15 min. 40 micrograms of total protein for each sample were separated by 8–10% SDS-PAGE and transferred onto a Westran S membrane (Whatman Inc. Floham Park, NJ). Desired proteins were probed with corresponding antibodies. Rabbit anti-human AKT, ERK, pAKT, pERK, EGFR, pEGFR, HER2 pHER2, HER3 and pHER3 antibodies (1:1000 dilutions) were purchased from Cell Signaling (Danvers, MA), mouse anti-human β-actin (1:10000 dilution) from Sigma (St. Louis, MO), anti-human EGFR and HER3 antibodies from Santa Cruz (Santa Cruz, CA), and anti-human pEGFR antibody from Millipore (Temcula CA). HRP-conjugated secondary anti-mouse and anti-rabbit IgG (H+L) was obtained from Promega (Madison, WI). Bound antibody was detected using the SuperSignal West Pico Chemoluminescence system (Pierce, Inc., Rockford, IL). Image J software was used for blot quantification. Protein densitometry was determined by Image J software.

Cells were treated with the two drugs and their combination with doses consistent with the cell proliferation assay for the indicated time. Cells lysates and xenograft tissue lysates were generated using lysis buffer as previously reported (27 (link)). The lysate was centrifuged at 16,000 g at 4°C for 10 min. 50 micrograms of total protein for each sample were separated by 10% SDS-PAGE and transferred onto a Westran S membrane (Whatman Inc. Floham Park, NJ). Desired proteins were probed with corresponding antibodies. Rabbit anti-human AKT, pAKT, pERK, pS6, and pHER3 (1:1000 dilutions) were purchased from Cell Signaling, mouse anti-human β-actin (1:10000 dilution) from Sigma (St. Louis, MO), anti-human EGFR and HER3 antibodies from Santa Cruz (Santa Cruz, CA), and anti-human pEGFR antibody from Millipore (Temcula CA). HRP-conjugated secondary anti-mouse and anti-rabbit IgG (H+L) was obtained from Promega (Madison, WI). Bound antibody was detected using the SuperSignal West Pico Chemoluminescence system (Pierce, Inc., Rockford, IL). Image J software was used for blot quantification.