Figure 2. HdrR and HdrM form stable heteromeric complexes.

A) Coimmunoprecipitation was used to detect heteromeric complex formation between HA epitope tagged HdrM (M-HA) and FLAG epitope tagged HdrR (R-FLAG). Samples were immunopurified with α-FLAG affinity resin and the results are shown using both α-FLAG and α-HA western blots. Elution samples were loaded in the same order as the input samples. B) The same HdrM-HA and HdrR-FLAG epitope tagged proteins were immunopurified with α-HA affinity resin. Elution samples were loaded in the same order as the input samples. C) Bimolecular fluorescence complementation (BiFC) with the yellow fluorescent protein (YFP) was used to detect the association of HdrR and HdrM in situ. Fluorescent images are shown above their corresponding light microscopy images. In the left panels, the N-terminal fragment of YFP was fused to HdrR (HdrR-N^YFP), while the C-terminal YFP fragment was simultaneously expressed as an unfused protein (C^YFP). In the middle panels, the N-terminal fragment of YFP was expressed as an unfused protein (N^YFP), while the C-terminal YFP fragment was simultaneously expressed as a fusion to HdrM (HdrM-C^YFP). In the right panels, the N-terminal fragment of YFP was expressed as fusion to HdrR (R-N^YFP), while the C-terminal fragment of YFP was simultaneously expressed as a fusion to HdrM (M-C^YFP). Images were captured using identical exposure times and camera settings. Scalebar indicates 1 μM. D) Tandem affinity coimmunoprecipitation in E. coli was used to detect heteromeric complex formation between heterologously expressed HdrR/M. A dual HA+FLAG epitope was fused to the C-terminus of HdrR to facilitate successive tandem affinity immunopurification of E. coli lysates using α-FLAG affinity resin immediately followed by a second immunopurification with α-HA affinity resin. Afterward, tandem affinity purified eluates were analyzed for the presence of HdrR/M via α-HA (HdrR-HA+FLAG) and α-GFP (HdrM-GFP) western blots. E. coli strains heterologously expressed different combinations of the following proteins: unmodified wild-type HdrR (HdrR), HdrM-GFP fusion (M-GFP), and dual epitope tagged HdrR (R-HAFLAG). Elution samples were loaded in the same order as the input samples.