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. 2016 Dec 27;292(6):2191–2202. doi: 10.1074/jbc.M116.736306

FIGURE 6.

FIGURE 6.

DOCK8 associates with LRAP35a through lobe B of the DOCK8 DHR2 domain. A, following the expression of WT DOCK8-HA or its mutants (DOCK8-ΔNΔDHR1-HA or DOCK8-ΔDHR2-HA) with LRAP35a-V5 in HEK-293T cells, cell extracts were immunoprecipitated with anti-HA antibody for immunoblotting. Data are representative of three independent experiments. B, following expression of GFP-tagged DOCK8-DHR2 (amino acid residues 1633–2071), DOCK8-DHR1 (amino acid residues 561–730), or GFP alone with LRAP35a-V5 in HEK-293T cells, cell extracts were immunoprecipitated (IP) with anti-GFP antibody for immunoblotting. Data are representative of three independent experiments. MW, molecular weight. C, recombinant protein encoding the His-SUMO-tagged DOCK8 DHR2 domain (20 μg) was incubated with GST alone, GST fusion LRAP35a, or its mutant (10 μg each) for pulldown assays. Data are representative of three independent experiments. CBB, Coomassie Brilliant Blue. D, following expression of GFP-tagged lobe A (amino acid residues 1633–1808), lobe B (amino acid residues 1764–1976), or lobe C (amino acid residues 1936–2076) of the DOCK8 DHR-2 domain with LRAP35a-V5 in HEK-293T cells, cell extracts were immunoprecipitated with anti-GFP antibody for immunoblotting. Data are representative of three independent experiments. E, following expression of GFP-tagged DOCK8-DHR2-lobe B, DOCK8-DHR1, or DOCK7-DHR2-lobe B (amino acid residues 1769–1981) with LRAP35a-V5 in HEK-293T cells, cell extracts were immunoprecipitated with anti-GFP antibody for immunoblotting. Data are representative of three independent experiments. F, following expression of WT DOCK8-HA or its VA mutant with LRAP35a-V5 in HEK-293T cells, cell extracts were immunoprecipitated with anti-HA antibody for immunoblotting. Data are representative of three independent experiments. G, following expression of WT DOCK8-HA or its VA mutant with LRAP35a-V5 in HEK-293T cells, cell extracts were pulled down with GST fusion Cdc42 or GST alone. Data are representative of three independent experiments.