Figure 3

PaSMC adherence to extracellular matrix regulates the MAP kinase pathway. Semiconfluent PaSMC cultured on collagen I, fibronectin and laminin and analyzed by Western blot or cold trypsin phosphorylation-specific flow cytometry to measure changes in pERK following stimulation with PMA and serum. A: Western blot analysis of pERK1/2 levels in PaSMC lysates from PMA and serum treated cells cultured on collagen I, fibronectin and laminin. Anti-smooth muscle actin is shown as a loading control. Normalized mean pixel intensity values for the pERK1/2 bands are indicated. B: Analysis of anti-pERK levels by cold trypsin phosphorylation-specific flow cytometry of PMA and serum treated PaSMC cultured on uncoated and collagen I, fibronectin and laminin-coated plates revealed a uniform increases in pERK activation. Plotted as overlaid histograms of fluorescence versus number of cells. C: Median fluorescence intensity (MFI) of anti-pERK levels determined by cold trypsin phosphorylation-specific flow cytometry of PMA and serum treated PaSMC cultured on collagen I, fibronectin and laminin. D: Plot of MFI values normalized to unstimulated PaSMC cultures on uncoated and collagen I, fibronectin and laminin-coated plates. Control: Goat-anti-rabbit Alexa Fluor 647 secondary only. (Representative of three independent experiments).