Pull-down assays and co-immunoprecipitation showing FtsZ-NDK interaction.

<p>(<b>A</b>) Pull-down assay using 6xHis-MtFtsZ and GST-MtNDK. Upper panel: GST-NDK is detected using anti-GST antibodies (mouse polyclonal). Lower panel: 6xHis-MtFtsZ is detected using anti-polyhistidine antibodies (mouse monoclonal). Lanes: 1. Ni²⁺-NTA agarose beads alone; 2. GST + 6xHis-MtFtsZ; 3. GST-MtNDK + 6xHis-MtFtsZ; 4. GST-MtNDK + 6xHis-MtFtsZ + anti-MtNDK antibodies (pre-incubated); 5. GST-MtNDK + 6xHis-MtFtsZ + anti-MtFtsZ antibodies (pre-incubated); 6. GST-MtNDK + 6xHis-MtFtsZ + anti-GST antibodies (pre-incubated). (<b>B</b>) Pull-down assay using 6xHis-EcFtsZ and GST-MtNDK. Upper panel: 6xHis-EcFtsZ is detected using anti-polyhistidine antibodies. Lower panel: GST-MtNDK is detected using anti-GST antibodies. Lanes: 1. GST-MtNDK + 6xHis-EcFtsZ; 2. GST-MtNDK + 6xHis-EcFtsZ + anti-MtNDK antibodies (pre-incubated); 3. GST-MtNDK + 6xHis-EcFtsZ + anti-EcFtsZ antibodies (pre-incubated); 4. GST-MtNDK + 6xHis-EcFtsZ + anti-GST antibodies (pre-incubated); 5. GST + 6xHis-EcFtsZ. Antibodies, whenever added to reaction mixtures were pre-incubated for 30 min at 4°C with the purified proteins at dilutions 1:100 for anti-MtNDK, anti-MtFtsZ, and anti-GST and 1:500 for anti-EcFtsZ before the addition of the cross-linker DTSP. The region where GST would have been on the blot is not shown. (<b>C</b>). Co-immunoprecipitation of recombinant MsNDK and MsFtsZ showing interaction. Lanes: 1–3. Co-IP with anti-MsNDK antibody; 4. Co-IP with anti-MsFtsZ antibody. Upper panel, western blotting with anti-MsFtsZ antibody; Lower panel, western blotting with anti-MsNDK antibody. Lanes: 1. MsNDK purified protein; 2. MsNDK, pre-incubated with anti-MsNDK antibody, followed by the addition of MsFtsZ; 3. MsNDK and MsFtsZ purified proteins; 4. MsFtsZ purified protein. (<b>D</b>) Co-immunoprecipitation of native MsNDK and MsFtsZ showing interaction in the total cell lysate. Lanes: 1. Purified recombinant 6x-His-MsNDK. 2. Protein A sepharose alone; 3. Protein A sepharose with <i>M</i>. <i>smegmatis</i> total cell lysate; 4. Co-IP with anti-MsNDK antibody; 5. Purified recombinant 6x-His-MsFtsZ; 6. Co-IP with anti-MsFtsZ antibody. Upper panel, western blotting with anti-MsFtsZ antibody; Lower panel, western blotting with anti-MsNDK antibody. Horizontal arrow heads indicates the endogenous MsNDK or MsFtsZ protein from the total cell lysate.