Fig. 2.

The ExbB dimer is a stable subunit of the ExbB tetramer. The parent strain for all of the plasmid variants (ΔexbBD ΔtolQRA), wild-type W3110 (WT), a strain expressing ExbB from the chromosome (ΔexbD ΔtolQR), and the plasmids expressing the ExbB variants are indicated above each lane of the immunoblot. Strains carrying exbB or exbD plasmids were expressed in the presence of 0.0005% l-arabinose (except for the N196A and G244C substitutions, where 0.001 and 0.002% l-arabinose were used, respectively). Mid-exponential-phase cultures were cross-linked with paraformaldehyde, transferred to LSB (23), and heated for 5 min at either 60 or 95°C. Samples were resolved on a 13% SDS-polyacrylamide gel and immunoblotted with anti-ExbB antibody. The positions of various ExbB complexes are indicated on the right. The arrowhead in the middle of lane N196A at 95°C indicates the position of the ExbB(N196A) tetramer. The positions of mass standards are on the left. A shorter exposure of the same immunoblot to indicate ExbB monomer levels is in the lower panel. Question marks (?) indicate unidentified protein complexes.