The effect of rPEDF mutants on ERK activation and proliferation in HUVECs. (A) HUVECs were serum starved for 16 hours and then stimulated with the indicated rPEDF mutants (10 nM) for 15 minutes. Cytosolic extracts (30 μg) were subjected to immunoblotting with anti-pERK (αpERK, top panel) or anti-gERK (αgERK, bottom panel) Abs. The positions of ERK2 and ERK1 are indicated. (B) Quantitative analysis of the results in panel A, presented as a mean ± SD (n = 3). ERK activation is indicated for both ERK1 and ERK2. (C) HUVECs were seeded in gelatin-coated 24-well plates in M-199 plus 2.5% or 5% FCS (0.5 mL/well). PEDFs were added immediately after seeding at quadruplicates (10 nM) with or without bFGF or VEGF (10 ng/mL each). After 48 hours, cell number was determined by methylene blue assay. The bar graph represents the mean ± SD (n = 5).