Fig 6.

GtrR is a substrate of Stk1 kinase. The cytoplasmic kinase domain of S. aureus Stk1 was expressed and purified as a GST-tagged fusion and incubated with purified His-tagged GtrR in the presence of the ATP analog ATPγS. Following PNBM alkylation, the labeled substrates were separated by SDS-PAGE, (A) coomassie stained, and immunoblotting used to detect (B) phosphorylated protein and (C) His-tagged GtrR. Phosphorylated protein and His-tagged GtrR were detected using an anti-thiophosphate ester and anti-His antibodies, respectively. An overlay of the His and thiophosphate ester signals is shown in (D). Bands corresponding to phosphorylated Stk1 and GtrR are annotated. (B–D) Raw images with noise were processed using noise reduction software to improve background clarity. Both the raw (top) and processed images (bottom) are shown for each immunoblot.