FIG 4.

Immunoblot analysis confirming the EloR-MltG interaction. Lysates from strains RH425 (wild type [wt]), ds515 (sfgfp-mltG), AW98 (flag-eloR), AW459 (flag-eloR hlpA-gfp), and AW447 (flag-eloR sfgfp-mltG) were incubated with resin beads tethered with anti-Flag antibodies to pull down Flag-EloR. As expected, immunoprecipitated Flag-EloR was found in strains AW98, AW459, and AW447 but not in strain ds515. sfGFP-MltG was found in immunoprecipitated fractions only when it was coexpressed with Flag-tagged EloR. The two EloR bands visible in the blot represent the phosphorylated and unphosphorylated forms of the protein (19). All fusion proteins used in the co-IP (HlpA-GFP, Flag-EloR, and GFP-MltG) have previously been shown to be stable when expressed in S. pneumoniae (19, 42). Uncropped versions of the immunoblots can be found in Fig. S6 in the supplemental material.