Figure 3 - Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner.

(a) Expression levels of mDia mRNAs during in vitro culture of TER119-negative mouse fetal erythroblasts, as quantified by quantitative real time PCR analysis. The relative level compared to 18S rRNA was calculated using the delta-delta Ct method. (b) Expression levels of mDia1 and mDia2 proteins during in vitro culture of TER119-negative mouse fetal erythroblasts analysed by western blot. (c) Glutathione-coated beads with bound GST or GST fusions with Rac1 or Rac2 were incubated with lysates of 293T cells that had been transfected with Flag-tagged mDia1 GBD or mDia2 GBD, together with GDP or GTPS as indicated. Beads were then subjected to western blotting using antibodies against the Flag tag. (d) Glutathione-coated beads with bound GST or GST fusions with indicated proteins were incubated with lysates of 293T cells that had been transfected with Flag-tagged mDia2 mutants as indicated, together with GTPS. Beads were then subjected to western blotting using antibodies against the Flag tag. (e) Glutathione-coated beads with bound GST or GST fusions with Rac1 or Rac2 were incubated with 1 g of purified mDia2 GBD produced in BL21 cells, together with GDP or GTPS as indicated. Beads were then subjected to western blotting using a polyclonal antibody against mDia2. The input of mDia2 GBD was shown by Coomassie staining. (f) Rac GTPases colocalize with mDia2 but not mDia1. TER119-negative mouse fetal erythroblasts were cultured as in Fig. 1 and harvested after 48 h in culture. Cells were fixed and stained with antibodies against Rac GTPases, mDia2 or mDia1. The scale bar represents 5 m. (g) Inhibition of Rac GTPases disrupts mDia2 localization at the CAR. TER119-negative mouse fetal erythroblasts were cultured as in Fig. 1. After 35 h in culture, 100 M NSC23766 was added to some cells and after 48 h in culture the erythroblasts were stained with Alexa Fluor 488–phalloidin for actin and antibodies against mDia2. The scale bar represents 7 m.