Fig. 5.

Nuclear localization of mDia2 was required for full activation of SMC-specific transcription. 10T1/2 cells were cotransfected with SM22-luciferase and the indicated mDia2 nuclear localization variant in the absence (A) or presence (B) of constitutively active L63RhoA. Luciferase activity was measured after 48 h and is expressed relative to empty expression vector (Ev) set to 1. *P < 0.05 vs. Wt. C: Cos7 cells were transfected with the indicated Flag-mDia2 variants. Lysates were incubated with glutathione S-transferase (GST) or GST-L63RhoA for 1 h. Following extensive washing, precipitates were run on an SDS page gel and probed with an anti-Flag Ab. D: 10T1/2 cells were cotransfected with SM22-luciferase and myocardin-related transcription factor (MRTF)-B in the presence or absence of the indicated mDia2 nuclear localization variants. E: quantification of GFP-MRTF-B localization in serum-starved 10T1/2 cells coexpressing the indicated mDia2 variants. At least 100 cells were counted under each condition from 3 independent experiments. *Nuclear localization P < 0.05 vs. empty vector. **Nuclear localization P < 0.05 vs. Wt. F: 10T1/2 cells were cotransfected with SM22-luciferase and FH1FH2 domain of mDia2 plus or minus MRTF-B. G: GFP-MRTF-B localization in serum-starved 10T1/2 cells in the absence (top) and presence (bottom) of the FH1FH2 domain of mDia2. Scale bar = 20 μm. H: 10T1/2 cells were cotransfected with the indicated luciferase reporter vector and either Wt mDia2 or an mDia2 variant in which the COOH-terminal NES was deleted (ΔNES). I: quantification of GFP-MRTF-B localization in 10T1/2 cells coexpressing Wt mDia2 or a mDia2 variant containing a mutation to the COOH-terminal NES (1064/5AA). At least 100 cells were counted under each condition from 3 independent experiments.