Figure 1: GEF-H1 translocates from microtubules to the cytoplasm and focal adhesions in response to LPA or thrombin.

(a) LPA or thrombin treatment disrupts GEF-H1–dynein complex. Serum-starved HEK293T cells, expressing Flag-tagged GEF-H1, were treated with LPA or thrombin for 5 min. Endogenous DIC, 14-3-3 and Tctex-1 proteins were detected in Flag-GEF-H1-precipitated complexes by western blot. Western blots of the whole-cell lysate (input) and immunoprecipitated complexes (IP) are shown. (b) LPA or thrombin induced GEF-H1 relocalization from microtubules. Confocal images of GEF-H1^−/−MEFs transfected with GEF-H1–eGFP were treated with LPA, thrombin or nocodazole, as indicated, for 5 min prior to fixation and staining with anti-tubulin. Scale bar, 20 μm. (c) Higher magnification (5 × 60) view of the images depicted in b. Scale bar, 20 μm. (d) The correlation coefficient (R) measuring co-localization between eGFP (GEF-H1) and polymerized microtubules in b was determined in 90 cells from three independent experiments. Error bars represent s.d. of at least three independent replicates, and P values derived from pairwise t-tests are indicated. (e) GEF-H1 translocates to focal adhesions in response to LPA. GEF-H1^−/−MEFs overexpressing GEF-H1–eGFP were treated with LPA for 1 h prior to fixation and staining with anti-vinculin antibodies (red) to visualize focal adhesions. Co-localization between GEF-H1 (GFP) and vinculin was measured with and without LPA treatment. Scale bar, 20 μm. (f) GEF-H1 relocalization using live-cell imaging. Images from movies of GEF-H1^−/− MEF cells transiently transfected with GEF-H1–eGFP and cherry tubulin, before and after LPA stimulation. Starved cells were treated with LPA. Data are representative of three independent biological replicates. Scale bar, 20 μm.