SLAIN2 links ch-TOG to EB1. (A) Mapping of the ch-TOG–binding domain of SLAIN1 and 2 (SL1 and SL2). CC, coiled coil; B/S, basic and serine rich; Y, C-terminal tyrosine. The ability to exert a dominant-negative (DN) effect on the number of EB1-positive MT tips is indicated. (B) Streptavidin pull-down assays were performed with extracts of HEK293 cells coexpressing BioGFP-SLAIN2 mutants and BirA and analyzed by Western blotting with the indicated antibodies. (C) Mapping of the SLAIN2-binding domain of ch-TOG. (D and E) GST pull-down assays were performed with the indicated GST fusions and lysates of HEK293 cells expressing GFP fusions of ch-TOG or SLAIN2. Coomassie-stained SDS-PAGE is shown for GST fusions. Western blots with anti-GFP antibodies are shown for GFP fusions and with ch-TOG antibodies for endogenous (Endog.) ch-TOG. White lines indicate that intervening lanes have been spliced out. (F and G) 3T3 (F) or HeLa cells (G) were transfected with GFP–SLAIN2-N1, fixed, and labeled with the indicated antibodies. In F, the insets show enlargements of the boxed areas where 1 is an untransfected control cell and 2 is a GFP–SLAIN2-N1–transfected cell. (H) Quantification of the number of EB1-positive comets per 100-µm² surface area in control or GFP–SLAIN1/2-N1–expressing HeLa cells. Approximately 10–50 cells were analyzed in each experiment. Error bars show SD. Statistically significant differences are indicated (**, P < 0.01; ***, P < 0.001). (I) A scheme of the identified interactions between SLAIN2, ch-TOG, EB1, CLASPs, and CLIP-170.