PKCβII activation by PMA increased assembly of focal adhesions.

<p>Scratch wounded IEC monolayers were allowed to migrate for 4 hours in the absence (control) or presence of PMA (PMA) and PKC isoforms inhibitors. The levels of total and phospo-paxillin were determined by immunoblotting (<b>A</b>) and quantified using densitometric analysis (<b>B</b>). The data are expressed as fold change relative to control after normalization to loading control. PMA treatment increased paxillin phosphorylation, which was attenuated by the inhibition of PKCβII (PMA+βII), but not PKCβI (PMA+βI). (<b>C</b>) Cell adhesion assay was used to examine IEC-matrix adhesion after PMA treatment in the presence or absence of specific inhibitors of PKC isoforms. PMA treatment significantly increased cell-matrix adhesion.This effect was reversed with inhibition of PKCβII, but not PKCβI. *significantly different (p<0.05). (<b>D</b>) To examine formation of focal adhesions by the cells at the leading edge (upper panels), and cells away from the wound (bottom panels) IECs were fixed and stained for F-actin (red) and phospho-paxillin (green). Representative images show increased phosphorylation of paxillin both at the lamellipodium and areas remote from the wound after PKC activation with PMA. The bar is 20 µm. (<b>E–F</b>) Quantification of the changes in the relative fluorescence intensity, as an indication of the changes in paxillin phosphorylation. (<b>E</b>) Average relative fluorescence intensity of 5 randomly placed lines (as shown in (D), bottom panels) per field of view, at least 7 fields per condition. N = 3 independent experiments. **significantly different (p<0.01). (<b>F</b>) Fluorescence intensity profiles of phospho-paxillin along representative white lines projected across fields of view (as shown on the images in (D), bottom panels) that were used for quantification in (E).