Fig. 4

Gefitinib inhibits phosphorylation of HER2 and HER3, but elevates HER2 and HER3 expression; HER2 and HER3 heterodimer associates with IKK and may be involved in Gefitinib-induced IKKβ/NF-κB signalling. a Gefitinib inhibits phosphorylation of HER2 and HER3 but induces levels of HER2 and HER3. Phosphorylation and total levels of HER2 and HER3 in the lysates from Fig. 3a were analysed by Western blot. b Knockdown of HER2 and HER3 abates Gefitinib-induced IKKβ/NF-κB signalling. Cal27 cells were transfected with non-targeting siRNA or siRNA against HER2 and HER3 for 24 h, and cells were treated with (DMSO) or Gefitinib (2, 5 or 10μmol/L) for 24 h. Phosphorylation status and total protein levels of IKKα/β and p65 were analysed by Western blot. c Cal27 cells were treated with increasing doses of Gefitinib for 4 h, lysed and immunoprecipitated with anti-HER2 or anti-HER3 antibodies. Lysates and immunoprecipitates were detected with HER2, HER3 and IKKβ as indicated by Western blot. d FaDu cells were treated with increasing doses of Gefitinib for 4 h and lysed. The cell lysates were immunoprecipated with anti-HER3 antibodies. Proteins in lysates and immunoprecitates were resolved by SDS-PAGE, and proteins were transferred to Pure Nitrocellulose Membrane (Bio-Rad), blocked in 5% nonfat milk, and blotted with the indicated antibodies after cutting the membrane as labelled (red lines)