FIGURE 7.
Analysis of GrlR2-GrlA complex formation by native gel electrophoresis. (A) GrlR2-GrlA protein complex formation in the enteropathogenic Escherichia coli (EPEC) strains grlR:3xFLAG, grlA:3xFLAG, ΔgrlR_grlA:3xFLAG, grlR:3xFLAG_ΔgrlA, as well as in the grlR:3xFLAG_ΔgrlA strain carrying plasmids pTEPGrlA1/I44A or pTEPGrlA1/R54A, was analyzed from samples taken from cultures grown in Dulbecco’s modified Eagle’s medium (DMEM) or Lysogeny Broth (LB) at an OD600 of 1. Total extracts were obtained from bacterial pellets and separated by 12% native PAGE. The GrlR2-GrlA and GrlR-GrlR complexes and the GrlR monomer (indicated by arrows) were identified by western blotting using anti-FLAG monoclonal antibodies. (B) The same cell extracts were also separated by 12% SDS-PAGE and transferred to a PVDF membrane for western blotting to confirm GrlR-3xFLAG and GrlA-3xFLAG expression in the different strains using anti-FLAG antibodies.