The effect of rPEDF, plPEDF, and the various rPEDF mutants on ERK/MAPK activation in HUVECs. (A) HUVECs were serum starved for 16 hours and then stimulated with different concentrations of rPEDF for the indicated times. 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) HUVECs were serum starved for 16 hours and then stimulated with rPEDF (10 nM) or with plPEDF (10 nM) for the indicated times. Cytosolic extracts (30 μg) were subjected to immunoblotting with anti–phospho ERK Ab (pERK, top panel) or with anti–general ERK Ab (gERK, bottom panel). (C-D) HUVECs were serum starved for 16 hours and then stimulated with rPEDF (10nM), plPEDF (10 nM), or with the various rPEDF mutants (10 nM) for 15 minutes. Cytosolic extracts (30 μg) were subjected to immunoblotting as described in panel A. (E) Quantitative analysis of immunoblots depicted in panels C-D is presented as a mean ± SD of 5 distinct experiments.