K853A and I704A FH1FH2mDia2 induce stable Glu MTs. (A) Glu and Tyr tubulin immunostaining of serum-starved NIH3T3 cells expressing indicated wild-type (WT) or actin mutant (I704A and K853A) EGFP-FH1FH2mDia2 constructs. (B) Quantification of cells injected with indicated constructs that exhibited stable Glu MTs (Glu). Data are mean ± SD from three independent experiments (n > 50 cells). Asterisks indicate P < 0.001, calculated by χ2 test (one degree of freedom). (C) Glu and Tyr tubulin immunostaining of serum-starved NIH3T3 cells expressing indicated constructs and then treated with 2 μM nocodazole for 30 min. (D) Quantification of cells that exhibited stable Glu MTs after injection of indicated constructs and nocodazole treatment as in C. Data are mean ± SD from three independent experiments (n > 50 cells). Asterisks indicate P < 0.001, calculated by χ2 test (one degree of freedom). (E) Western blot analysis of pulldown experiments using GST-EB1, GST-APC-C, or GST alone incubated with the indicated His-FH1FH2mDia2 proteins. Bound proteins were eluted and resolved by SDS-PAGE and mDia2 proteins were detected by mouse RGS-His antibody. Bars, 10 μm.
