The SH3 domain determines recruitment of IRTKS to RAB11.
A, schematic of MIM, IRTKS, and their fusion mutants. WH2, WASP homology 2. B, HeLa cells expressing GFP-tagged IRTKS or IRTKSΔSH3 proteins were stimulated with 150 ng/ml SDF-1 for 30 min. The cell lysates were subjected to immunoprecipitation (IP) with GFP antibody, followed by immunoblot (IB) using anti-RAB11, RAB5, or RAB7, as indicated. C, cells expressing GFP-tagged MIM, IRTKS, and IRTKSΔSH3 were treated with 500 ng/ml SDF-1 for up to 45 min. The levels of surface CXCR4 were estimated by flow cytometry. The data represent mean ± S.E.M. (n = 3). *, p < 0.05; ***, p < 0.001; referring to the differences between cells expressing IRTKS-GFP and those expressing IRTKSΔSH3-GFP. D, cells expressing MIM-GFP or its fusions were treated with 150 ng/ml SDF-1 for 30 min. The interactions of MIM and its fusions with RAB7 and RAB11 were analyzed as described in B.