Figure 3

Muropeptides binding to PrkC and its effect on kinase activity in vitro. (A) Coomassie-stained SDS-PAGE of partial proteolysis profile of the full-length PrkC (left) or its catalytic domain, PrkCc, used as negative control (right). 3 µg of PrkC and PrkCc were incubated with trypsin (Promega) in the absence or presence of increasing amounts of TCT (0, 50, 100, 150 and 250 µM) for 10 min at 37 °C. The digestion profiles were assessed by electrophoresis in SDS-PAGE. For full-length PrkC (left gel), the arrow indicates a band whose intensity decreases in the presence of increasing amounts of TCT. The area where the pattern of digestion is modified by the addition of TCT is framed and magnified under the full-length gel. (B) In vitro phosphorylation assays in the presence of PrkC or PrkCc. 2.5 µg of protein were incubated with [γ-³³P]ATP and MBP and increasing amounts of TCT (0, 100, 150 and 250 µM) at 37 °C during 15 min. Samples were separated by SDS-PAGE and visualized by autoradiography. Full-length gels are presented in the supplemental data.