Fig 5.

Fyn regulates PIKE-A/STAT5a interaction. (A) Fyn activation enhances PIKE-A/STAT5a association. HEK293 cells were transfected with different combinations of plasmids as indicated. Transfected PIKE-A was precipitated using GST pulldown, and the associated STAT5a was detected using anti-GFP antibody (1st panel). Expressions of the transfected STAT5a (2nd panel), PIKE-A (3rd panel), and Fyn (4th panel) are also shown. (B) Mutation of Fyn phosphorylation sites in PIKE-A abolishes its binding with STAT5a. HEK293 cells were transfected with different combinations of plasmids. The association between PIKE-A and STAT5a was examined using GST pulldown (1st panel). Expressions of the transfected STAT5a (2nd panel), PIKE-A (3rd panel), GST (4th panel), and Fyn (5th panel) were also verified. (C) Inhibition of Fyn diminishes the PIKE-A/STAT5a association. HEK293 cells were cotransfected with GFP-STAT5a and mGST-PIKE-A. The transfected cells were then treated with PP2 (1 μM) or PP3 (1 μM) for 24 h. The interaction between PIKE-A and STAT5a was then examined using immunoprecipitation (top panel). Expressions of the transfected STAT5a (middle panel) and PIKE-A (bottom panel) were also verified. Relative values of the coprecipitated STAT5a after normalization with the expression of mGST-PIKE-A are indicated at the bottom of top panel. (D) Reduced PIKE-A/STAT5a interaction in Fyn^−/− WAT. Endogenous PIKE-A in the inguinal WAT of wild-type and Fyn^−/− (3-month-old) mice was immunoprecipitated using antibody against the C terminus of PIKE-A, and the associated STAT5a was examined (1st panel). Total tyrosine phosphorylation of the immunoprecipitated PIKE-A was also determined (3rd panel). Expressions of the endogenous STAT5a (2nd panel), PIKE-A (4th panel), and Fyn (5th panel) are also shown.