Rab5 and the GTPase-deficient mutant, Rab5Q79L, lead to SUN2 redistribution to endosomes.
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(A) Left: SUN2 coimmunoprecipitated with Rab5, using antibodies against c-myc and Rab5, in the protein lysate from HeLa cells cotransfected with pcDNA3-HA-SUN2-myc and pEGFP-Rab5. Middle: No binding between SUN2 and EGFP was observed in cells overexpressing HA-SUN2-myc and EGFP. Right: The antibody against c-myc could not recognize any bands in HeLa cells transfected with pEGFP-Rab5 only. Molecular weight is shown in kDa. (B) Immunofluorescence staining reveals that endogenous SUN2 partially colocalized with transfected EGFP-Rab5 in endosomes (arrows). (C) In the cell expressing pEGFP-Rab5Q79L, SUN2 was relocated to the enlarged endosomes from the NE (arrow), but not in Rab5Q79L-negative cells (arrowheads). (D) In Rab5S34N-positive cells, SUN2 remained on the NE (arrow). Scale bar, 10 µm. (E) Antibodies against HA and Rab5 were used as precipitating/immunoblotting antibodies in HeLa cells cotransfected with plasmids abbreviated on the top. HA-SUN2 coimmunoprecipitated with wild type or mutant forms of EGFP-Rab5. In the control shown on the right, the antibody against HA did not immunoprecipitate Rab5 in HeLa cells transfected with pEGFP-Rab5 only. IB, immunoblotting; IP, immunoprecipitation.