Figure 3. Phosphorylation of Y416 requires kinase activity and is sustained upon suppression of activity.

AD293 cells were transfected with the indicated constructs and analysed by Western Blot 24 h post-transfection. Blots were performed at least twice, which showed consistent results; a single representative is shown. Key: KD, kinase dead K293 M mutant; Em, Emerald fluorescent protein; closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src; β-gal, β -galactosidase in same vector as c-Src constructs. A. Western Blot standardized for c-Src levels. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. B. Western Blot standardized for STAT3 levels.