Signal transducer and activator of transcription 5b tyrosine phosphorylation and transcriptional activation. (a) Mouse embryo fibroblast (MEF5-/-) cells were transfected with signal transducer and activator of transcription 5b (STAT5b) along with pRc (vector), breast tumor kinase (Brk), K219M, or Y447F. Immunoprecipitations (IPs) were performed using antibodies specifically against STAT5b and were analyzed by immunoblotting with antibodies directed toward phospho-Y699 STAT5b (top) or STAT5b (bottom). (b) MEF5-/- cells were transfected with various STAT5b constructs along with constitutively active Y447F Brk. Immunoprecipitations (IPs) were performed as above and were analyzed via immunoblotting with antiphosphotyrosine antibody (top) or anti-STAT5b antibody (bottom). (c) MEF5-/- were transfected with the STAT5-specific Spi2.1 promoter luciferase construct and either His-vector, His-STAT5b, or His-Y699F with or without Brk. Luciferase activity was measured and normalized to total protein. Values from four independent experiments performed in triplicate reported as the fold induction over His, pRc ± standard error: His, pRc (1.00 ± 0.00) and Brk (1.05 ± 0.39); STAT5b, pRc (28.89 ± 5.51) and Brk (75.48 ± 25.79); Y699F, pRc (1.17 ± 0.14) and Brk (0.93 ± 0.27). Student's t test was used to determine statistical significance between STAT5b pRc and STAT5b Brk. *P = 0.0123.