Figure 4.

GidA and MnmE interact with each other. (A) Western blot of GST pull down analysis using anti-GidA antibody. GST-MnmE or GST alone were used as baits for pull down analysis. Input: total lysate of strain IC5332 that was added to the corresponding bait previously immobilized to glutathione-agarose. Sup: fraction not retained by the resin-bound bait protein. El: the bound material that is eluted from the resin with glutathione buffer. Equivalent amounts of each fraction were loaded. (B) The same as in (A), but the baits were GST-GidA or GST, the lysate was from strain IC5287, and the western blot was developed with anti-MnmE antibody. (C) Coimmunopurification of FLAG-GidA and MnmE. FLAG-GidA was purified from strain DH5α/pIC1180 with anti-FLAG agarose as described in Materials and Methods, and the resulting fractions were analyzed by western blot using the antibodies indicated in the right side of each panel. Anti-EF-Tu was used as a negative control. The arrowhead marks MnmE position. The upper band detected in the MnmE western blot corresponds to an unrelated, unknown protein that is recognized by the anti-MnmE antibody and serves as a negative control. CL: cleared lysate of DH5α/pIC1180 (supernatant after sonication). Ins: pellet of cleared lysate. FT: flow through after incubation with anti-FLAG agarose. R: resin after washes. El 1: first eluate with elution buffer (free FLAG peptide). El 2: second eluate with elution buffer. (D) In vitro pull down analysis of purified recombinant MnmE (rMnmE) and FLAG-GidA proteins. After incubating both proteins together, anti-FLAG resin was added and fractions collected as described in Material and Methods. As a negative control, MnmE was processed without adding FLAG-GidA. Sup: supernatant after pelleting FLAG-agarose. El: proteins eluted with free FLAG peptide. Equivalent amounts of samples were loaded in each well. Size of molecular weight markers (MWM) is indicated on the left in kDa.