Figure 6.

Mitotic phosphorylation disrupts SLAIN2 interaction with MTs, EB1, and ch-TOG. (A) Proportion of mitotic HeLa cells identified by staining with antibodies against histone H3 phosphorylated at serine 10 after transfection with the indicated siRNAs. (B) HeLa cells stably expressing GFP-SLAIN2 (SL2) were fixed and stained with anti-EB1 antibodies. The prophase cell was distinguished by the strong increase in centrosomal MT nucleation. Insets show enlargements of the boxed areas. (C) IPs with anti-SLAIN1/2 antibodies from control HeLa cells or cells blocked in mitosis with 0.1 µM nocodazole. Where indicated, immunoprecipitated material was treated on beads with λ phosphatase. Western blotting was performed with the indicated antibodies. (D) The same experiment as in C but using a GFP-SLAIN2 stable HeLa cell line and anti-GFP antibodies. (E) GST pull-down assays were performed with GST or GST-EB1, and cell extracts were prepared as in D. (F) The same experiment as in C but using HeLa cells transiently expressing N- and C-terminal fragments of SLAIN2. (G and H) HeLa cells stably expressing GFP-SLAIN2 were blocked in mitosis with 7.5 µM STLC (G) or 0.1 µM nocodazole (H) and released for 1 h into medium containing either the indicated inhibitors, 20 mM NaCl, or 20 mM LiCl. White lines indicate that intervening lanes have been spliced out. S, STLC; N, nocodazole; FP, flavopiridol; Endog., endogenous; Cont., control; Mit., mitotic; Extr., extract.