Fig. 7.

MMG8 binds to EB1 and mediates its recruitment to the Golgi. (A) Immunoprecipitation of MMG8 was performed and the immunoprecipitates (IPs) and inputs were immunoblotted with the indicated antibodies. (B) MMG8 contains the SxIP (x denotes any amino acid residue) motif that is required for interaction with EB1; residues highlighted in black are crucial for EB1 binding (asterisks). Pongo abelii MMG8, NP_001126198; mouse MMG8, NP_835181. (C) MMG8 and its L311A/P312A mutant (311/2A) were ectopically expressed. After immunoprecipitating the proteins by targeting the tag (anti-FLAG IP), the immunoprecipitates and inputs were immunoblotted. Vector, FLAG vector; WT, wild-type MMG8. (D) HEK293T cells were doubly transfected with GFP–EB3 and the MMG8 constructs. The immunoprecipitates and inputs were immunoblotted (IB) for EB3 (anti-GFP) and MMG8 (anti-FLAG). (E,F) HeLa cells transfected with the indicated siRNAs were triple-stained for MMG8, EB1 and TGN46. The cells were either left untreated (E) or were extracted with a saponin-containing buffer (F) before fixation. The boxed areas are enlarged on the right. (G) Cells transfected with the indicated siRNAs were treated with nocodazole to depolymerize microtubules, after which the cells were immunostained. The boxed areas are enlarged on the right. The images shown for all immunofluorescence experiments represent >90% of ∼500 cells analyzed for each condition. Scale bars: 5 µm.