FIG. 5.
Analyses of the KinA protein complex by BN-PAGE and cross-linking. (A) Strains coexpressing GFP-tagged full-length KinA or KinAN and FLAG-tagged full-length KinA, both under the control of Phyper-spank, were constructed and examined by a coimmunoprecipitation assay. Cell extracts were prepared from strains MF3373 (KinA-FLAG and KinA-GFP) (lane 1) and MF3375 (KinA-FLAG and KinAN-GFP) (lane 2) grown in LB medium in the presence of IPTG (10 μM). The extracts were then processed for coimmunoprecipitation as described in the legend to Fig. 4. Samples of immunoprecipitates were separated by BN-PAGE as described in Materials and Methods. GFP-tagged full-length KinA and KinAN were detected with anti-GFP antibodies. A native protein marker kit (Invitrogen) served as molecular mass standards (lane M). Possible combinations of complex formation are listed, along with the molecular mass of each tagged protein. The individual monomeric forms were not detected, but the sizes are indicated for convenience. (B) Cell extracts were prepared from strains MF3352 (full-length KinA-GFP) (lanes F) and MF3360 (KinAN-GFP) (lanes N) grown in LB medium in the presence of IPTG (10 μM). The extracts were then processed for the BMH cross-linking reaction as described in Materials and Methods. Samples were separated by SDS-PAGE (10%), followed by immunoblot analysis with anti-GFP antibodies. −, no BMH; +, with BMH. Possible combinations of complex formation are listed, along with the molecular mass of each tagged protein.