SLAIN1 and SLAIN2 are EB-dependent +TIPs. (A and C) GST pull-down assays were performed with the indicated GST fusions and lysates of cells expressing different GFP-SLAIN1/2 fusions. Coomassie-stained gels are shown for GST fusions, and Western blots with anti-GFP antibodies are shown for GFP fusions. (B) Mapping of the minimal MT plus end–binding domain of SLAIN2 based on GST-EB1 binding and MT plus end tracking in live cells. CC, coiled coil; B/S, basic and serine rich; Y, C-terminal tyrosine. Asterisks indicate SxIP-like motifs. Mutations in the SxIP-like sites are indicated by red asterisks (see Fig. S1 C for the sequence of SLAIN2). (D) Live-cell imaging of HeLa cells transiently transfected with EB3–monomeric RFP and GFP-SLAIN1/2. Red and green images were collected simultaneously with a beam splitter and a 0.5-s interval; five consecutive frames were averaged. (E) IPs from HeLa cell extracts with rat monoclonal antibodies against HA tag (control) or EB1 and EB3 were analyzed by Western blotting with the indicated antibodies. White lines indicate that intervening lanes have been spliced out. (F and H) 3T3 cells were transfected with the indicated siRNAs, fixed, and stained with the indicated antibodies. The insets show enlargements of the boxed areas. In the overlay, EB1 is shown in green, and SLAIN2 is shown in red. (G) Extracts of 3T3 cells transfected with the indicated siRNAs analyzed by Western blotting with the indicated antibodies. Endog., endogenous; SL2, SLAIN2; WB, Western blot.