Figure 6

In vitro degradation assays to determine MecA-mediated proteolysis of SigX. A. A colorimetrical assay of ATPase activity of ClpC. The reactions were initiated in a buffer by adding the following protein(s): (1) ClpC alone (black stars), (2) ClpC and MecA (open squires), (3) ClpC and SigX (black triangles), and (4) ClpC, MecA and SigX (black circles). These proteins were treated with PreSciession protease to remove GST-tag before used for the assay. B. The degradation reactions were initiated in a reaction buffer, including Group 1: ClpC-His, MecA-His, SigX-His, GST-ClpP and ATP (lane 1 and 2), Group 2: ClpC-His, MecA-His, SigX-His and GST-ClpP without addition of ATP, and Group 3: ClpC-His, SigX-His, GST-ClpP and ATP without MecA-His (lane 5 and 6). Aliquots of samples were taken from the reactions to assess degradation results by Western blot analysis of the interacting proteins using the anti-His or anti-GST antibody. C. Degradation assay of the reaction mixture containing ClpC, MecA, SigX and ClpP after removal of the GST-tag by PreScission protease cleavage. The SigX was detected by Western blotting using the anti-SigX antibody and the remaining SigX protein on the membrane was scanned and converted as RIDV values.