FIG. 4.

Interaction of Runx2 with SRF. (A) 293T cells were transiently transfected with HA-SRF or FLAG-Runx2 alone or together. Cell lysates were incubated with anti-HA or anti-FLAG antibodies or normal mouse IgG, and immunoprecipitates (IP) were subjected to SDS-PAGE and immunoblotted with anti-FLAG or anti-HA antibodies as indicated. Whole-cell extracts were loaded for input. (B) GST pull-down assay shows that Runx2 directly interacts with SRF. In vitro-translated ³⁵S-labeled SRF was incubated with either GST alone or GST-Runx2. After vigorous washing, bound protein and 10% input were detected by autoradiography after SDS-PAGE. A reciprocal experiment was performed using in vitro-translated ³⁵S-labeled Runx2 and GST-SRF. (C) C3H10T1/2 whole-cell extract was immunoprecipitated with anti-SRF antibody or normal rabbit IgG, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-Runx2 and anti-SRF antibodies. (D) In vitro-translated ³⁵S-labeled myocardin or HERP1 was incubated with either GST alone or GST-Runx2. Interacting proteins were visualized by autoradiography after SDS-PAGE. (E and F) Upper panels show schematic representations of deletion constructs of GST-Runx2 (1 to 4) and HA-SRF (1 to 4) used in the respective lower panels. Lower panels show the mapping of the interactive domain of Runx2 and SRF by a GST pull-down experiment. (E) In vitro-translated ³⁵S-labeled full-length SRF and deletion mutants of GST-Runx2 were incubated. (F) A reciprocal experiment was performed using in vitro-translated ³⁵S-labeled HA-SRF fragments and GST-Runx2. After vigorous washing, bound protein and 10% input were subjected to SDS-PAGE and Coomassie brilliant blue (CBB) staining and then detected by autoradiography.