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

FIGURE 4.

FIGURE 4.

DOCK8 regulates macrophage migration through LRAP35a interaction. A, following the expression of DOCK8-HA, βPIX-HA, MRCKα-GFP, and/or LRAP35a-V5 in HEK-293T cells, cell extracts were immunoprecipitated (IP) with anti-HA antibody or anti-GFP antibody for immunoblotting. Data are representative of at least three independent experiments. MW, molecular weight. B, cell extracts from BM-derived macrophages were pulled down with GST alone or GST-fusion LRAP35a, and the bound proteins and total lysate control were probed by immunoblot with anti-DOCK8 antibody. The recombinant proteins used in this assay were detected with Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments. C, following the expression of DOCK8-HA or MRCKα-GFP in HEK-293T cells, cell extracts were pulled down with GST fusion LRAP35a or its mutants. Data are representative of three independent experiments. D, RT-PCR analysis for the expression of Lurap1 encoding LRAP35a in BM-derived macrophages transfected with siRNA or control oligonucleotide. E, effect of LRAP35a knockdown on the migration speed of Dock8+/− BM-derived macrophages. As controls, Dock8+/− and Dock8−/− BM-derived macrophages transfected with control oligonucleotide were analyzed simultaneously. At least 40 cells were analyzed per category. The central lines and error bars on the scatterplots represent the mean and S.D., respectively. **, p < 0.01 (two-tailed Mann-Whitney test). Data were collected from three separate experiments. F, effect of expression of the LRAP35a N mutant on the migration speed of Dock8+/− BM-derived macrophages. As controls, Dock8+/− and Dock8−/− BM-derived macrophages transfected with GFP alone were analyzed simultaneously. At least 20 cells were analyzed per category. The central lines and error bars on the scatterplots represent the mean and S.D., respectively. **, p < 0.01 (two-tailed Student's t test). Data were collected from five separate experiments. G, effect of LRAP35a knockdown on CCL2-induced MLC2 phosphorylation at Ser-19 (phospho-MLC) in Dock8+/− BM-derived macrophages. As controls, Dock8+/− and Dock8−/− BM-derived macrophages transfected with control oligonucleotide were analyzed simultaneously. Results were quantified by densitometry and are expressed as the ratio of the phosphorylated form to total protein after normalization of the 2-min value of control samples to an arbitrary value of 1. Data are indicated as the mean ± S.D. of four independent experiments. *, p < 0.05 (two-tailed Mann-Whitney test). H, effect of MRCKα knockdown on CCL2-induced MLC2 phosphorylation at Ser-19 (phospho-MLC) in Dock8+/− BM-derived macrophages. As controls, Dock8+/− and Dock8−/− BM-derived macrophages transfected with control oligonucleotide were analyzed simultaneously. Results were quantified by densitometry and are expressed as the ratio of the phosphorylated form to total protein after normalization of the 2-min value of control samples to an arbitrary value of 1. Data are indicated as the mean ± S.D. of four independent experiments. *, p < 0.05 (two-tailed Mann-Whitney test).