FIG 3.

Immunoblot analysis of STAT3 phosphorylations. (A) Serum-starved EC were treated with EGF, TRAP, or both for 15 min, and STAT3 was immunoprecipitated and resolved by SDS-PAGE. STAT3 phosphorylations were assessed by immunoblotting. The anti-pThr⁷¹⁴ antibody was generated by immunizing a rabbit with a phosphorylated peptide corresponding to phosphorylation at that site (KFICVpTPTTC). STAT3 Ser⁷²⁷ phosphorylation was detected using a Pro-Met-phospho-Ser-Pro motif antibody and a commercially available antibody from Santa Cruz Biotechnology. (B) Densitometry analysis of Thr⁷¹⁴ phosphorylation from 3 independent experiments performed as for panel A. (C) Densitometry analysis of Ser⁷²⁷ phosphorylation from 3 independent experiments performed as for panel A. (D) Serum-starved EC were treated with EGF at the indicated concentrations in the presence of TRAP for 15 min. The EC were lysed with RIPA buffer, and STAT3 was immunoprecipitated and resolved by SDS-PAGE. Three antibodies (Ab) were used to assess Tyr⁷⁰⁵ phosphorylation. Ab 1 is Cell Signaling Technology (CST) 9131, Ab 2 is CST 9145, and Ab 3 is CST 9138. The immunoblot with CST 9138 was deliberately overexposed to film to detect basal STAT3 Tyr⁷⁰⁵ phosphorylation. Thr⁷¹⁴ and Ser⁷²⁷ phosphorylation was assessed as for panel A. IL-6 (10 ng/ml) was included as a positive control for Tyr⁷⁰⁵ phosphorylation. The error bars indicate SEM.