In vitro Y699 phosphorylation by breast tumor kinase. In vitro Y699 phosphorylation of signal transducer and activator of transcription 5b (STAT5b) by breast tumor kinase (Brk). Purified recombinant GST, GST-STAT5b, or GST-Y699F were incubated with (+) or without (-) purified recombinant Brk in reaction buffer containing 20 nM ATP at 30°C for 30 minutes. An equal volume of 2 × Laemmli buffer was added to end the reaction and the samples were run on SDS-PAGE. Gels were analyzed via (a) silver stain and (b) by immunoblotting with an antiphospho-Y699 STAT5b-specific antibody.