Cetuximab-resistant (CtxR) clones have increased HER family receptor activity. (A) CtxR cells have increased HER family activity as well as phosphorylation levels of downstream signaling molecules. Parental (HP) and CtxR clones (HC1, HC4, HC8) were harvested and protein lysates were fractionated on SDS-PAGE followed by Immunoblotting for the indicated proteins. α-Tubulin was used as a loading control. (B) Expression of EGFR and HER3 on the plasma membrane is increased in CtxR clones compared to cetuximab-sensitive (CtxS) cells (HP). The levels of EGFR and HER3 on the cell surface were measured by flow cytometry. (n = 3) (C) EGFR displayed increased association with HER3 in all three CtxR clones compared to CtxS HP cell line. Cells were harvested and EGFR or HER3 were immunoprecipitated with anti-rabbit EGFR antibody or anti-rabbit HER3 antibody. The immunoprecipitate complexes were fractionated on SDS-PAGE followed by immunoblotting for indicated proteins.