Fig. 5. Interaction between Rab5a GTP and syntaxin-4 on neutrophil phagosomes during phagocytosis. Neutrophils ingested H37Rv, H37Ra or S. aureus for the indicated periods of time, after which the proteins associated with the isolated phagosomes (750 µg of protein for each phagosome fraction) were solubilized and Rab5a or syntaxin-4 was immunoprecipitated as described in the Materials and Methods. The immunoprecipitates (IP) were separated by SDS-PAGE transferred and blotted on the membranes. The GTP-binding state of Rab5a was detected on MCPv, MCPa and SCP by an [-³²P] GTP overlay assay and visualized by autoradiography. The same membrane was stripped and analysed for the presence of syntaxin-4. Interaction of GTP-bound Rab5a and syntaxin-4 on the MCPv (A) and MCPa (B) during 120 minutes and SCP (C) during 60 minutes of phagocytosis are shown. The immunoblots are representative of three separate experiments. (D) The kinetics of association of Rab5a-GTP on the MCPv () and on SCP ([UNK]) and that of the synatxin-4 on the MCPv () and SCP () are illustrated. Data are expressed as mean±s.e.m of three experiments. (E) Syntaxin-4 immunoprecipitated from the MCPv, MCPa and SCP fractions isolated after 30 minutes phagocytosis and Rab5a-GTP co-immunoprecipitated with anti-syntaxin-4. The blot is representative of three independent experiments.

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