CHICA binds to DYNLL1 via TQT consensus motifs. (A) HEK293T cells were transfected with full-length and deletion constructs of GFP-CHICA for 30 h. CHICA complexes were immunoprecipitated using sheep anti-GFP antibodies, and then Western blotted using mouse anti-GFP or rabbit anti-DYNLL1 antibodies. (B) HEK293T cells were transfected with Myc-CHICA, and single or combined TQT to AAA mutants for 30 h. CHICA complexes were immunoprecipitated using mouse anti-Myc antibodies, and then Western blotted using rabbit anti-Myc, sheep anti-HMMR, or rabbit anti-DYNLL1 antibodies. (C) HeLa cells stably expressing GFP-DYNLL1 were treated with control siRNA or directed toward the 3′-UTR of CHICA for 72 h, and transfected with the wild-type or 3x TQT to AAA mutant forms of Myc-CHICA, fixed after 36 h, and then stained as indicated in the figure. DYNLL1 was directly visualized by GFP fluorescence. Bar, 10 µm. (D) The percentage of cells with aligned or rotated spindles is plotted in the bar graph. Error bars show the SEM (n = 30 in three experiments).