Figure 4. Heterodimerization and auto-regulation by MucR1/2.

(a) Immunoblots showing that MucR2 co-purifies with MucR1-TAP before (that is, in cell lysates, lysate) or after the first TAP purification step (cleavage with TEV protease, TAP). The upper panel shows a blot probed with antibodies to MucR1 (α-R1). The asterisk indicates untagged MucR1 in wild-type (NA1000) cells. The arrowheads indicate MucR1-TAP before (black) or after (red) cleavage of the protein-A moiety in the TAP tag with the TEV protease, eluting the proteins from the IgG agarose beads. The two lowest panels shows a blot probed with antibodies to MucR2 (α-R2) revealed at two different exposures. The lower amount of MucR2 that co-purifies with WT MucR1-TAP compared with MucR1(Y97C) or MucR1^Tn5 proteins is likely because of a reduction in the total MucR2 levels when MucR1 is overexpressed. Molecular size standards are indicated on the left as blue lines with the corresponding values (blue) in kDa. (b) Traces of the occupancy of various transcription factors at the ctrA promoter based on ChIP-seq data. (c) β-Galactosidase measurements in extracts of WT (dark blue) and ΔR1/2 (light blue) cells harbouring a P_ctrA-, P_mucR1- or P_mucR2-lacZ promoter-probe plasmid. Data are from three biological replicates. Error is shown as s.d. (d) Electrophoretic mobility shift assay (EMSA) showing the binding of His₆-SUMO-MucR1 or -MucR2 to P_ctrA. The white arrows indicate the unbound probe. The black arrows indicate the shifted ctrA probes bound by MucR1. His₆-SUMO-MucR2 or His₆-SUMO does not retard the probe.