Fig. 4. Involvement of host protein tyrosine phosphorylation in the internalization of Rickettsia conorii into Vero cells. (A,B) Immunofluorescence microscopy of infected cells demonstrates that extracellular R. conorii recruits tyrosine phosphoproteins (pTyr) to sites of adherence within 15 minutes of infection (arrows point to bacteria that have unambiguously recruited tyrosine phosphoproteins). (C) Specificity of pTyr protein colocalization with adherent R. conorii is not caused by antibody crossreactivity to bacterial antigens (arrowheads). (D) Pre-incubation of Vero cells with a PTK inhibitor, genistein, blocks R. conorii entry in a concentration-dependent fashion, highlighting a role for PTKs in bacterial entry. (E) Immunoprecipitation and western immunoblot analysis using anti-phosphotyrosine antisera (pTyr) revealed that R. conorii infection of Vero cells induces the phosphorylation of host proteins (arrows) 10 minutes after infection, with strong phosphorylation of a p125/130 protein. Immunoblot analysis of pre-immune precipitated cellular lysates with anti-actin antisera serves as a protein loading control. Bar, 3 µm.