MICAL1 connects GRAF1b/2 to Rab8 and Rab10. (A) Immunoprecipitation (IP) of transfected 293T cells with α-GFP. The efficiency of uMICAL1 binding was quantified using coimmunoprecipitation with GFP-Rab8a as reference. n = 2–5. MICAL1 bound to Rab8a, Rab8b, and Rab10. (B) Confocal images of transfected HeLa cells. GFP-MICAL1 colocalized with RFP-Rab8a, Rab8b, and Rab10 but not Rab1 or Rab11a. (C and D) Confocal images of transfected HeLa cells stained with α-Rab8 or α-MICAL1. (C) Endogenous Rab8 was found on uMICAL1 tubules. (D) Endogenous MICAL1 was found on GFP-Rab8a tubules. (E and F) shControl or shMICAL1-expressing cells were transfected with RFP-Rab8a or Rab10. (E) Total skeletal length of RFP-Rab8a/10 structures per cell. n = 22–36 cells. (F) Proportion of cells with Rab8a/10–positive tubules. n = 3. (G–I) Immunoprecipitation (IP) of transfected 293T cells with α-myc–coated beads (G and H) or α-myc (I). (G and H) myc-MICAL1^tail only coimmunoprecipitated GFP-Rab8a; myc-MICAL1^Pro only coimmunoprecipitated GRAF2-GFP. (I) GRAF2-GFP was only coimmunoprecipitated with myc-Rab8a when GFP-MICAL1 was coexpressed, not GFP-MICAL1 PPAPP. (A, E, and F) Data are means ± SEM; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. (B–D) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.