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. 2019 May 14;20(8):1119–1133. doi: 10.1111/mpp.12818

Figure 5.

Figure 5

Flp regulates HrpX by interacting with the promoter sequence of hrpX. (A) Electrophoretic mobility shift assays (EMSA) to examine the interaction between the hrpX promoter sequence and Flp protein. In this experiment, DNA is acquired from purified PCR products of the hrpX promoter sequence with 5ʹ‐FAM labelled primers. The DNA is then incubated with purified Flp protein (protein final density 0, 0.25, 0.5, 0.75, 1 and 1.5 µM) and the hrpX promoter DNA is 0.10 µmol). A competition assay was also conducted with unlabelled DNA. When the concentration of unlabelled DNA increased from 10 to 30 µM, the Flp‐DNA complexity decreased, indicating the unlabelled DNA of hrpX brings competition to the 5'‐FAM‐labelled hrpX in forming DNA–protein complex with Flp. F, free DNA strips; R, retarded DNA strips. (B) In vitro transcription experiment using Flp protein (final density arranged from 0, 0.1, 0.20 and 0.5 µM) in the transcription system. A template DNA fragment containing the clp promoter (Liu et al., 2019) and a 121‐bp hrpX fragment extending from +1 to +121 relative to the TIS were used as controls.