Fig. 4.3.

PLA activity of PEDF-R. Detergent-soluble membrane fractions from R28 cells were assayed for PLA activity using (1,2-dilinoleoyl)-phosphatidylcholine as substrate and lipoxygenase as the coupling enzyme in reaction buffer (50 mM Tris-HCl, pH 7.5, 3 mM deoxycholate) as described (Jimenez-Atienzar et al. 2003). Formation of the product was measured spectrophotometrically by increase in the absorbance at 234 nm per min (dA/min; y-axis). (a) Dose response of PLA activity of R28-derived PEDF-R. Proteins from R28 cell membranes were solubilized with phosphate buffered saline pH 6.5 containing 0.1% NP-40, and the PLA activity was determined for increasing amounts of detergent-soluble protein fractions, (b) Effects of PEDF on the R28-derived PEDF-R activity. Extracts were preincubated with PEDF for 10 min and then assayed for PLA activity. Concentrations of PEDF used in each assay are indicated in the x-axis. (c) PEDF-R proteins were solubilized from ARPE-19 membrane protein precipitate with phosphate buffered saline pH 6.5 containing 0.5% CHAPS in the absence (grey boxes) or presence of 0.8 μM PEDF (black boxes). PLA activity was measured as in Panel B in the absence (none) or presence of 10 nM PEDF