FIG. 4.

GST pulldown assays for Cdt protein interactions. Whole-cell lysates of E. coli expressing cdt operons encoding M45 epitope-tagged CdtA, CdtB, or CdtC proteins (as indicated to the left of each panel) were probed with purified GST-CdtB, GST-CdtA, or GST (as a negative control) as indicated below each panel. Cdt proteins in the samples before (Pre) and after (Post) the pulldowns or bound to the glutathione-agarose beads (pellet) were detected by Western blotting with a monoclonal antibody directed to the M45 epitope tag. The levels of CdtB in E. coli lysates were not sufficient for its detection in the pre- or postpulldown samples without prior concentration. However, precipitation with GST-CdtA resulted in the concentration of CdtB, which allowed its detection in the sample.