2.5. Electrophoretic Mobility Shift Assay (EMSA)

EMSA was performed as described previously [24]. Briefly, Cy3-labeled DNA fragments used as probes in EMSA were generated by PCR using pCRC656, pCRC659, and pCRC660, containing the ldhA promoter region with or without the mutated GlxR-binding site as a template. GlxR was incubated with cAMP prior to incubation with the labeled probe. The resulting DNA–protein complexes were loaded onto a 5% polyacrylamide gel and visualized using a Typhoon TRIO Variable Mode Imager (GE Healthcare Bioscience).