Figure 4.
PELP1 regulates STAT3 Ser727 phosphorylation via the src-MAPK pathway. A, COS-1 cells were cotransfected with or without PELP1 and treated with EGF for 15 minutes. The total lysates were immunoblotted with a phospho-MAPK–specific antibody. B, MCF-7 cells stably expressing pcDNA or PELP1 were treated with EGF for various periods of time, and MAPK activation was analyzed by Western blot analysis with a phosphospecific antibody. C, MCF-7 cells stably expressing pcDNA or PELP1 were pretreated with or without the MAPK inhibitor PD98059 for 60 minutes and then treated with EGF for 15 minutes. MAPK activation was analyzed by Western blotting. D, MCF-7 cells were cotransfected with the STAT3 luciferase reporter gene with or without PELP1. The cells were pretreated with or without PD98059 and stimulated with EGF for 8 hours. The STAT3 reporter gene activity was determined. E, MCF-7 cells stably expressing pcDNA or PELP1 were pretreated with or without the src inhibitor PP2 for 60 minutes and then treated with EGF for 15 minutes. The status of MAPK activation and STAT3 Ser727 phosphorylation was analyzed by immunoblotting. F, murine fibroblast cells deficient in src kinase (SYF cells) were transfected with or without PELP1 and treated with or without EGF, and STAT Ser727 phosphorylation was measured by immunoblotting.