FIG 2.

CrhB-CTH can interact with AnaRne directly. (A) Schematic of the domain composition of CrhB according to the alignment. The numbers indicate the amino acid positions. (B) Far-Western blotting assay was conducted to evaluate the interaction between recombinant AnaRne and CrhB (or the truncated variants). The PVDF membrane represented on the left was incubated with skim milk as a control, the one in the middle was incubated with SAnaRne, and the one on the right was incubated with SAnaRne and MNase. SAnaRne and BSA served as a positive control and a negative control, respectively. Other proteins were purified with the C-terminal His tag. “FW” represents far-Western blotting. The final results were detected with the anti-strep antibody. The amount of protein used here was 1 μg, with the exception that 20 ng was loaded for SAnaRne. (C) Bacterial two-hybrid analysis of the interactions of CrhB (or its truncated variants) and AnaRne. “Rne” denotes AnaRne, “CrhBN” represents CrhB-NTH, and “CrhBC” stands for CrhB-CTH. The horizontal rows represent the E. coli BTH101 cells carrying the recombinant “T18” protein fusion plasmids, whereas the vertical columns represent the cells carrying the recombinant “T25” protein fusion plasmids. These results were based on three independent experiments.