Fig. 4.

Anti-cEGFR p527 antibodies block EGFR and HER2 signaling and in vitro growth. cEGFR p527 immune serum blocks the signaling of EGF induced EGFR phosphorylation and HER2 signaling as assessed by pERK immunoblot. GAPDH was used as a protein loading control. For growth inhibition, cells were incubated with 1:50 or 1:100 dilutions of immune dog serum. Cetuximab served as the positive control for A431 cells, Ab-1 was the positive control for D17 cells and trastuzumab was the positive control for MDA-MB-453 cells. Cell proliferation was measured by ³H-thymidine incorporation and growth inhibition relative to preimmune serum, calculated as described in the Materials and Methods. A. Inhibition of EGF signaling in D17 cells. B. Ratio of pEGFR/GAPDH in D17 cells as determined by densitometry of immunoblots. C. Inhibition of D17 growth. D. Inhibition of EGF signaling in A431 cells. E. Ratio of pEGFR/GAPDH in A431 cells as determined by densitometry of immunoblots. F. Inhibition of A431 growth. G. Inhibition of HER2 signaling in MDA-MB-453 cells. H. Ratio of pERK/GAPDH in MDA-MB-453 cells by densitometry of immunoblots. I. Inhibition of MDA-MB-453 growth.