MICAL1 G3W, WDR44 ΔC, and GRAF1/2 BAR-PH are dominant negative mutants interfering with Rab8/10–mediated trafficking and WDR44 tubulation. (A) Confocal images of transfected HeLa cells. GFP–MICAL1 G3W colocalized with GRAF1b-RFP; GFP–MICAL1 ΔMO colocalized with RFP-tagged GRAF2, GRAF1b, and Rab8a on intracellular puncta and tubules. (B) Confocal images of transfected HeLa cells stained with α-GRAF2. Endogenous GRAF2 was found on GFP–MICAL1 ΔMOΔCH puncta and tubules. GFP–MICAL1 ΔMOΔCH had a lower nuclear background than GFP–MICAL1 ΔMO. (C) Immunoprecipitation (IP) of transfected 293T cells with α-myc. GFP-tagged Rab8a, Rab10, GRAF2, and GRAF1b were coimmunoprecipitated by myc–MICAL1 G3W as well as by myc-MICAL1. (D) Confocal images of transfected HeLa cells. RFP-WDR44 did not colocalize with GFP–MICAL1 G3W. (E) Confocal images of HeLa cells cotransfected with GFP–WDR44 ΔC and RFP-VAMP3 and incubated with Alexa Fluor 647–Transferrin (10 µg/ml, 1 h). The three proteins colocalized on intracellular peripheral patches. (F) Confocal images of transfected HeLa cells stained with α-WDR44 and α-MICAL1. Unlike MICAL1, endogenous WDR44 was found on GRAF1 BAR-PH–GFP puncta and tubules. (G) Confocal images of transfected HeLa cells. GRAF1 BAR-PH–GFP colocalized with RFP-tagged Rab8a and Rab10 on intracellular tubules and puncta. (A, B, and D–G) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.