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. 2000 Sep 26;97(20):10884–10889. doi: 10.1073/pnas.97.20.10884

Figure 5.

Figure 5

Accumulation of a mixed disulfide between DsbA and DsbB in dsbB R48 mutants under aerobic conditions. Proteins of cells growing exponentially in M63 medium were AMS-alkylated, separated by native SDS/PAGE, and subjected to Western blotting with anti-DsbA serum (lanes 1–8) or purified DsbB antibody (lanes 9–16). HK205 (wild type; lanes 1, 7, 9, and 15), HK207 (dsbB C41Y; lanes 2 and 10), HK209 (dsbB R48C; lanes 3 and 11), HK211 (dsbB R48H; lanes 4 and 12), HK227 (dsbB∷Kan; lanes 5, 8, 13, and 16), and HK229 (dsbA∷Kan; lanes 6 and 14). Cells contain plasmid pHP18 carrying dsbA C33Y in lanes 7, 8, 15, and 16. ∗, DsbA–DsbB complex; oxi, the oxidized form of DsbA; red, the reduced form of DsbA; arrowhead, DsbA C33Y; arrows, a nonspecific crossreacting band. Positions of molecular mass standards are indicated on the left. The migration of mixed-disulfide complexes differs slightly reflecting differences in the number of free cysteine residues in each complex: DsbA–DsbBR48C (+1), DsbA–DsbBR48H (0), DsbAC33Y–DsbB (−1).