Figure 4

Shear stress decreased DAPK phosphorylation at serine 308 on glass and membrane substrates. (A) Western blot of overall DAPK expression in control and sheared cells (6 hours). Loading control bands are probed with anti-actin antibody on the same blot. DAPK expression increased with extended shear (Top). Quantitative analysis of band intensities was carried out on at least 4 separate sample sets. DAPK expression relative to actin is presented in the panel below as fold increase over control cells, for both glass and membrane samples. For both glass and membrane, DAPK expression increased with extended shear (Bottom). Data represent average ± standard error (n = 4) with * P < 0.01 compared to control-glass cells and + P < 0.05 compared to control-membrane cells. (B) Western blot of phosphorylated DAPK at serine 308, for control and sheared cells (6 hours). Loading control bands are probed with anti-actin antibody on the same blot (Top). Quantitative analysis of phospho-serine DAPK expression as fold increase over control, showed a decrease in phosphorylated DAPK serine 308 with 6hr shear, for both glass and membrane substrates (Bottom). Data represent average ± standard error (n = 4) with * P < 0.01 compared to control-glass cells and + P < 0.05 compared to control-membrane cells. (C) Phosphorylated DAPK as a fraction of total DAPK expression was calculated for each sample group based Western blot analysis. Compared to control cells, the fraction of phosphorylated DAPK decreased to a minimum of 38% for glass and 43% for membrane after 6 hours of shear stress. Data represent average ± standard error for 4 independent experiments (n = 4). # P < 0.01 compared to control-glass and Δ P < 0.01 compared to control-membrane.