Fig. 5
PAK and ERK activation in response to MCP-1 is dependent on GPCR βγ subunit, PI3Kγ, and ROCK activity. a Immunoblotting for phosphorylation of PAK1Ser199/204 and PAK2Ser192/197 and total PAK1/2. Serum starvation of BMSCs in combination with 10 μM RS102895, 10 μM MPS-Phos, 5 μM LY294002, or 10 μM Y27632 for 24 h and 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 Densitometric quantification of a (PAK1Ser199/204 and PAK2Ser192/197 phosphorylation). Untreated serum-starved BMSC PAK1Ser199/204 and PAK2Ser192/197 levels were set to 1. Graph represents three separate independent experiments. Data are presented as mean ± standard deviation. c Immunoblotting for phosphorylation of ERK1/2 and total ERK1/2 following serum-starved BMSCS was pre-treated with MCP-1 for the indicated times or with 10 μM RS102895, 10 μM MPS-Phos, 5 μM LY294002, or 10 μM Y27632 and exposed to MCP-1; 100 μg of total cell extract/lane. Representative blot of three separate independent experiments is shown. d Densitometric quantification of c (ERK1/2 phosphorylation). Untreated serum-starved BMSC pERK1/2 levels were set to 1. Data are presented as mean ± standard deviation. Graph represents three separate independent experiments. BMSC bone marrow-derived stromal cell, ERK extracellular signal-regulated kinase, GPCR G protein-coupled receptor, MCP-1 monocyte chemoattractant protein 1, MPS-Phos MPS-phosducin-like protein C terminus, PAK P21 protein (Cdc42/Rac)-activated kinase 1, PI3K PI3 kinase, ROCK Rho-associated, coiled-coil containing protein kinase