FIG. 4.

The BfpB complex can be chemically cross-linked with BfpG. (A) BfpG is a monomer under conditions that BfpB is a multimer. Three micrograms of outer-membrane protein from the B171-8 wild-type strain was incubated in sample buffer containing 4% SDS for 7 min at various temperatures. Following SDS-PAGE, Western blot analysis using αBfpG antiserum was performed. BfpG runs as a monomer at every incubation temperature. (B) DTSSP cross-links BfpG to the BfpB multimer. One milligram of outer membranes from the wild-type B171-8 strain was treated with a 0.2 mM solution of the reducible cross-linker DTSSP. After cross-linking, BfpB complexes were purified. The same two adjacent sucrose gradient fractions, containing BfpB complexes, were used in each of the following immunoblots. Lanes 1 and 2, anti-BfpB immunoblot of purified complexes incubated at 22°C in sample buffer with no 2-ME; lanes 3 and 4, anti-BfpB immunoblot of purified complexes incubated at 100°C in sample buffer with no 2-ME; lanes 5 and 6, anti-BfpG immunoblot of purified complexes incubated at 100°C in sample buffer with no 2-ME; lanes 7 and 8, anti-BfpG immunoblot of purified complexes incubated at 100°C in sample buffer with 2-ME. The intensity of the cross-linked complex decreases and BfpG monomer appears.