Quantitative analysis of the effect of palmitoylation on cell surface expression of PAR2.

<p><i>A</i>, Anti-PAR2 N19 antibody flow cytometry analysis of non-permeabilised CHO-PAR2-myc and CHO-PAR2-C361A-myc cells treated with either DMSO (negative control) or 2BP for 16 h. Secondary antibody only MFI values were subtracted from N19 values and these are shown relative to values from CHO-vector untreated cells. Values were determined from 3 independent experiments and are shown as Mean ± SD. <i>B</i>, (Left panel) Examination of plasma membranePAR2 levels by cell surface biotinylation. Live cells were reacted with membrane impermeant EZ-link NHS-SS-biotin. Plasma membrane (PM) and intracellular (IC) fractions were collected from whole cell lysates and subjected to anti-myc, anti-GAPDH (control for IC fraction) and anti-β1 integrin (control for PM fraction) Western blot analysis. The data are representative of 3 independent experiments. (Right panel) Graphical representation of densitometric analysis of data from these 3 experiments. Values of PM PAR2 were normalised to β1 integrin signal and are displayed as Mean ± SD. <i>C</i>, Anti-PAR2 N19 antibody flow cytometry analysis of DU145 cells transiently transfected with vector, PAR2-GFP or PAR2-C361A-GFP. MFI values from 3 independent experiments were used to calculate fold change of cell surface PAR2 and PAR2-C361A relative to vector transfected cells and are displayed as Mean ± SD. **, <i>P</i><0.05; ***, <i>P</i><0.001.