Fig. 3

SS2 infection of hBMEC induces the ligand-dependent transactivation as well as the dimerization of EGFR. a Total RNAs extracted from hBMEC were analyzed by real-time PCR for the transcription of ErbB family genes. GAPDH was used as an internal control for normalization. b Immunoblot analysis of ErbB family members in whole cell extracts after infection of hBMEC with SC19 at an MOI of 10 for the indicated times. β-actin in cell lysates was analyzed as normalization control. c, d EGFR or ErbB3 was immunoprecipitated and immunoblotted with an anti-phosphotyrosine antibody (4G10) and then with the anti-EGFR or anti-ErbB3 antibody (top and middle). After stripping, the blots were reprobed with anti-ErbB3 or anti-EGFR antibodies to determine the possible interaction of EGFR with ErbB3. e Determination of ErbB3 expression in hBMEC transfected with either vehicle control or ErbB3 shRNA. f Knocking down of ErbB3 via shRNA attenuated SS2-induced EGFR activation. g AG1478 treatment significantly inhibited SS2-stimulated ErbB3 activation in hBMEC. h Heat-inactivated SC19 was unable to induce transactivation of EGFR, compared with the time-dependent activation of EGFR in response to viable SS2. i The induction of EGFR-related ligands in hBMEC in response to viable or heat-inactivated SS2 were compared by real-time PCR. GAPDH was used as the internal control. j Pretreatment with batimastat decreased SS2-induced transactivation of EGFR