Figure 1.

Substrate specificity and subcellular localization of ARHGEF10. (A) The domain structure of ARHGEF10. Amino acid residue numbers are shown. DH, Dbl homology; PH, pleckstrin homology. (B) GEF activity of ARHGEF10 toward RhoA. HA × 3-tagged wild-type (wt) or constitutively activated (G14V) RhoA was expressed with Myc-tagged Dbl or Myc-tagged ARHGEF10(376–665) in COS7 cells. Levels of the GTP-bound form were measured by pull-down assays using GST-Dia(1–304). Expression levels of RhoA and GEFs were determined by immunostaining with anti-tag antibodies. (C) Co-localization of ARHGEF10 and RhoA in centrosomes. HA × 3-tagged RhoA(wt) was expressed in HeLa cells, and indicated proteins were immunostained. Scale bar, 10 μm. (D) Subcellular localization of ARHGEF10 in unsynchronized cells. HeLa cells were cultivated in the growth medium, and indicated proteins were immunostained. Scale bar, 10 μm. (E) Subcellular localization of ARHGEF10 in G1/S phase. HeLa cells were synchronized in G1/S phase with thymidine, and indicated proteins were immunostained. The high magnification image of the boxed area in the 3rd panel is shown in the 4th panel. Scale bar, 10 μm (1st-3rd panels) and 2 μm (the 4th panel). Flow cytometric analysis of DNA content is also shown. (F) Subcellular localization of ARHGEF10 in M phase. HeLa cells were synchronized in M phase with nocodazole, and indicated proteins were immunostained. The high magnification image of the boxed area in the 3rd panel is shown in the 4th panel. Scale bar, 10 μm (1st-3rd panels) and 2 μm (the 4th panel). Flow cytometric analysis of DNA content is also shown. In (C) to (F), nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and arrowheads indicate centrosomes. Thin dotted lines in cell images in (C) to (F) indicate the contour of the cell.