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. 2017 Dec 13;8:2502. doi: 10.3389/fmicb.2017.02502

FIGURE 7.

FIGURE 7

Electrophoretic mobility shift assay (EMSA) analysis of SinR binding to the PkinB region carrying deletion or base substitutions that affect β-Gal synthesis on lacZ-fusion analysis. (Top) The nt sequence of the PkinB region (nt –75/+10) is shown, SinR-1 and SinR-2 being indicated. (A) [1] EMSA results for SinR-binding to the PkinB (–75/+10) probe in a 5% non-denaturing polyacrylamide gel. Increasing amounts of SinR (0, 12.5, 25, 50 nM) were used. (nM was calculated as the SinR monomer.) Upper and lower bands resulting from SinR-binding to the probe appeared. [2,3,4] EMSA results using the PkinB (–75/+10) probe carrying the Δ5 deletion, the G-45A base substitution, and Δ5 and G-45A, respectively. Hence, these mutant probes carry only an intact SinR-2. The arrowheads [2,4] indicate the position of the shifted band resulting from SinR binding to SinR-2. (B) EMSA results with the PkinB (-55/+10) probe carrying only SinR-2 consisting of C2-1 and C2-2. [1] EMSA results using the PkinB (–55/+10) probe (wild-type). [2] EMSA results using the PkinB (–55/+10) probe carrying the T-27C C-26T substitution in the C2-1 consensus sequence. DNA probes were prepared by use of primer pair and template DNA listed in Supplementary Tables S1, S2-2.