Figure 5
From: The guanine nucleotide exchange factor Arhgef7/βPix promotes axon formation upstream of TC10
Arhgef7 interacts with TC10. (a) Bacterially expressed GST-TC10 was coupled to glutathione-sepharose beads and incubated with lysates of HEK 293T cells transfected with the expression vector for HA-Arhgef7. Bound Arhgef7 was analyzed by Western blot using an anti-HA antibody. The expression of comparable amounts of GST proteins was visualized by Coomassie blue staining. (b) Schematic representation of Arhgef7 domains expressed as GFP fusion proteins that were used for pull-down experiments. (c) Bacterially expressed GST or GST-TC10 was coupled to glutathione-sepharose beads and incubated with lysates of HEK 293T cells transfected with the expression vectors for GFP-Arhgef7, or GFP-fusion proteins for the CH, CH-SH3 of DH-PH domains as indicated. Bound GFP-fusion proteins and the expression of comparable amounts of protein was analyzed by Western blot using an anti-GFP antibody or Coomassie blue staining. Molecular weights are indicated in kDa. (d) Homology models obtained for a complex between Arhgef7 (cyan) and TC10 (green) are shown with Arhgef7 depicted in cyan and TC10 in green. Putative polar interactions between Arhgef7 and TC10 are represented by broken yellow lines connecting alpha-carbon atoms of each residue. (e) Bacterially expressed GST or GST-PBD that specifically binds active TC10 was coupled to glutathione-sepharose beads and incubated with lysates of HEK 293T cells transfected with the expression vectors for GFP-TC10 or GFP and HA-Arhgef7 (+) or pcDNA3.1-HA (−) in the presence (+) or absence (−) of a phosphatase inhibitor as indicated. Bound TC10 and the expression of comparable amounts of protein were analyzed by Western blot using anti-HA and anti-GFP antibodies or Coomassie Blue staining. Molecular weights are indicated in kDa.
