Tyrosine phosphatase SHP2 is activated by ProS and is involved in the inhibitory activity of ProS on VEGF-A–mediated EC proliferation. (A) Changes in EC BrdU incorporation, in response to the indicated factors and effects of SHP2 inhibitor NSC87877 (100μM). ECs were seeded at a density of 6 × 103 cells/well in 96-well plates in growth factors containing medium for 24 hours, switched to a growth factor–depleted medium containing the indicated factors at the specified concentrations. BrdU incorporation was measured by ELISA. (B) Western blot analysis of MAPK-Erk1/2 activation state after pretreatment with NSC 87877 (100μM) for 3 hours before ProS stimulation for 15 minutes followed by VEGF-A stimulation for 5 minutes. (C) Intensity of bands was quantified and is represented as a percentage of control of the ratio of phosphorylated Erk1/2 over Erk1/2. (D) Western blot analysis of Akt activation state after pretreatment with NSC 87877 (100μM) for 3 hours before ProS stimulation for 15 minutes followed by VEGF-A stimulation for 5 minutes. (E) Intensity of bands was quantified and is represented as a percentage of control of the ratio of phosphorylated Akt over Akt. (F) Western blotting analysis of SHP2 phosphorylation subsequently to EC culture exposure to human ProS. Subconfluent EC cultures were exposed in a growth factor–depleted medium to 10 μg/mL human ProS or vehicle for 15 minutes. Cell cultures were then lysed, and equal amounts of proteins were analyzed by Western blotting using either an anti–phospho SHP2 or anti-SHP2 antibody. (G) Intensity of bands was quantified and is represented as a percentage of control of the ratio of phosphorylated SHP2 over SHP2. (H) Western blot analysis of MAPK-Erk1/2 activation state after treatment with ProS (10 μg/mL) for the indicated time. (I) Intensity of bands was quantified and is represented as a percentage of control of the ratio of phosphorylated Erk1/2 over Erk1/2. (J) Western blot analysis of Akt activation state after treatment with ProS (10 μg/mL) for the indicated time. (K) Intensity of bands was quantified and is represented as a percentage of control of the ratio of phosphorylated Akt over Akt. (A-K) Data were obtained from 3 independent experiments (3 independent cell culture) in either 96-well plates (A) or 24-well plates (B-K), each either in triplicate wells (A-G) or in duplicates (H-K), expressed as percentages of control ± SD. ***P < .001. **P < .01. *P < .05. ns indicates not significant.
