mDia1 and mDia2 form hetero-oligomers.

(A) 293T cells were transiently transfected with constructs expressing FLAG-mDia2 with or without HA-mDia1. The cell lysates were collected and immunoprecipitated with anti-HA (upper panel) or anti-FLAG (lower panel) magnetic microbeads. The immunoprecipitants were subjected to Western blotting assays with the indicated antibodies. (B) Immunoprecipitation assay in 293T cells overexpressing HA-mDia1 (left) or mDia2-3×FLAG (right). Western blotting analyses with indicated antibodies following the indicated co-immunoprecipitation assays were shown. (C) Co-immunoprecipitation assays were performed using anti-mDia1 (left) or anti-mDia2 (right) antibodies in 293T cell lysates followed by Western blotting of the indicated proteins. (D) Schematic map showing wild-type mDia2 domains and different truncated mutants. (E) Co-immunoprecipitation of HA-tagged mDia1 full length with indicated mDia2 full length or truncated mutants tagged by C-terminal 3 ×FLAG. The lysates were extracted from 293T cells transfected with indicated constructs, and immunoprecipitation assays were performed using anti-FLAG magnetic beads. The retained proteins on the beads were blotted and visualized by indicated antibodies. (F) Schematic diagram of wild-type mDia1 domains and different truncated mutants. (G) Co-immunoprecipitation of mDia2-3×FLAG with HA-tagged mDia1 full-length or truncated mutants by using anti-HA magnetic beads. The interacted proteins remaining on the beads were examined by Western blotting using indicated antibodies. (H) 293T cells stably expressing HA-mDia1 were transfected with empty vectors or constructs encoding mDia2 full length or DAD tagged with 3×FLAG at the C-terminus. Anti-FLAG immunoprecipitation was performed followed by immunoblotting. (I) Purified GST-mDia2 full-length and truncated mutants (DAD and FH1-FH2) were incubated with His-mDia1 full-length or GBD-DID. Proteins retained on agarose beads after GST pull-down were blotted with the indicated antibodies. The purified GST or GST fusion proteins were examined by Coomassie blue staining. (J) 293T cells were transiently co-transfected with indicated constructs for 48 hours. The cells were then fixed and subjected to immunofluorescent staining with anti-HA (Red) and anti-FLAG (Green) antibodies. Scale bar, 10μm. The asterisks indicate the specific target protein bands. Error bars represent the SEM of the mean. * p<0.05, ** p<0.01. Two-tailed unpaired student’s t-test was used to generate the p values.