Combined treatment of CtxRclones with cetuximab and U3-1287 inhibits HER2, AKT and MAPK signalings more effectively than either drug alone. (A) Human Phospho-Kinase array analysis demonstrated that combined treatment with cetuximab and U3-1287 inhibits proliferation and survival signaling in CtxR cell clone, HC4. The cell extracts were incubated with membranes containing antibodies to 46 different kinase phosphorylation sites. Quantitation of phosphorylated proteins was completed using scanned images from ImageJ software. Data points are represented as the mean of duplicate spots. (B) Effects of combined cetuximab and U3-1287 treatment on their respective kinase targets in CtxR clones. Protein lysates from Figure 5A (HC4) were fractionated on SDS–PAGE followed by immunoblotting for the indicated proteins. Protein lysate from other CtxR clones (HC1 and HC8) as well as HP cells were obtained after treatment with vehicle, cetuximab (20 ug/mL), U3-1287 (100 ug/mL) or the combination of cetuximab and U3-1287 for 24 h. α-Tubulin was used as a loading control.