Figure 1

Subcellular distribution of wild-type and mutant Rab24 expressed in 293 cells. (A) Cells were transfected with vectors encoding mycRab24wt or mycRab24(D123I) and 24 h later the expressed proteins were localized by immunofluorescence microscopy, using primary antibodies against the myc epitope or the Rab24 C-terminal hypervariable domain, as indicated above each panel. At the exposure setting used for the photograph, the Rab24 antibody did not give a detectable signal in the non-transfected cells (indicated by asterisk). The bar equals10 microns. (B) Cells were transfected with a vector encoding Rab24(D123I) without the myc epitope, and the expressed protein was localized with the Rab24 antibody. (C) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in buffer without detergent and fractionated by centrifugation as described in the Methods. The cytosol and particulate fractions were subjected to SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody to detect the expressed proteins. (D) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in high-detergent buffer (see Methods) and the proteins recovered in the detergent-soluble and insoluble fractions were assayed by SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody.