Fig. 4

MCP-1-induced migration is dependent on GPCR βγ subunit and PI3K activity. a Kinetics of MCP-1-mediated AKT signalling. Immunoblotting for phosphorylated Akt^Ser473 and total Akt following serum-starved BMSCs exposed to MCP-1 for the indicated times; 100 μg of total cell extract/lane. Representative blot of three separate independent experiments is shown. b Dependence of the MCP-1 signalling cascade on CCR2 and PI3Kγ activity (inhibited by 24-h serum starvation combined with 10 μM RS102895 and 10 μM LY294002, respectively). Immunoblotting for pAkt^Ser473 and total Akt. Whole cell extracts prepared from serum-starved BMSCs pre-treated with the indicated inhibitors (10 μM RS102895, GPCR βγ subunit inhibitor 10 μM MPS-Phos, or 5 μM PI3K inhibitor LY294002) for 24 h prior to MCP-1 exposure for the indicated time; 100 μg of total cell extract/lane. Representative blot of three separate independent experiments is shown. c Densitometric quantification of b (Akt^Ser473 phosphorylation). Untreated serum-starved BMSC Akt^Ser473 levels were set to 1. Graph represents three separate independent experiments. Data are presented as mean ± standard deviation. d PI3Kγ activity is required for MCP-1-induced BMSC migration in three dimensions. PI3Kγ activity inhibited by 24-h pre-treatment with 5 μM LY294002. Graph represents three separate independent experiments. Data are presented as mean ± standard deviation. A P value of less than 0.05 (*) was deemed significant. BMSC bone marrow-derived stromal cell, CCR2 chemokine (C motif) receptor 2, GPCR G protein-coupled receptor, MCP-1 monocyte chemoattractant protein 1, PI3K PI3 kinase, SFM serum-free medium