Figure 1 - Sequence of FilGAP and its interaction with FLNa.

(a) The primary sequence of FilGAP. The pleckstrin homology (PH), RhoGAP and coiled-coil domains are underlined in order of appearance. (b) A schematic diagram of FilGAP and its homologue domain structures. Residues phosphorylated by ROCK in vitro are indicated as -P. (c) GST-fusion filamin A (FLNa) fragments shown in the diagram, or GST alone, were incubated with FilGAP protein (residues 552–749), and precipitated with glutathione–Sepharose beads. Bound FilGAP proteins were analysed by western blotting using anti-FilGAP antibody. Each GST construct is shown after Coomassie (CBB) staining. (d) GST fusion FilGAP fragments, or GST alone, were incubated with FLNa protein, and precipitated with glutathione–Sepharose beads. Bound FLNa proteins were analysed by western blotting using an anti-FLNa antibody. Each GST construct is shown after Coomassie staining. (e) Endogenous FLNa associates with recombinant FilGAP in cells. cDNAs encoding HA-tagged full-length FilGAP or mutant FilGAP lacking FLNa-binding domain (CC) were transfected in HEK293 cells. FilGAPs were immunoprecipitated from cell extracts using anti-HA antibody, and bound FLNa was identified by western blotting using anti-FLNa antibody. (f) Endogenous FilGAP associates with endogenous FLNa. FilGAP protein was immunoprecipitated from HEK cell extracts using anti-FilGAP antibody and the bound FLNa was identified by using anti-FLNa antibody. Full scans for e and f are shown in the Supplementary Information, Fig. S5. IgG, immunoglobulin G.