Fig. 2.

PolD1 inhibits γTuRC-induced microtubule nucleation in vitro. a The recombinant protein FLAG-PolD1 was tested for binding with purified γTuRCs. FLAG-PolD1 was absent in the control. After anti-FLAG immunoprecipitation, the input γTuRCs and the immunoprecipitates were immunoblotted. b Purified γTuRCs reconstituted with the γTuRC stimulator CDK5RAP2(51–200) were used in the microtubule-nucleating assay together with PolD1 at various concentrations. Representative microscopic fields of polymerized microtubules are shown. The data are presented as means ± s.e.m. of three independent experiments. c Purified γTuRCs were used for microtubule nucleation in the absence of CDK5RAP2(51–200). PolD1 wild-type and mutants (D515V and S605del) and PolD2 were added at an excessive amount (356 nM). Shown are representative images of polymerized microtubules, and the data are presented as means ± s.d. of three independent experiments. Scale bars, 10 μm