FIG. 4.

YfgL residue S172 makes direct contact with YaeT. (A) Cells producing YfgL-His₆ (YfgL_6His) harboring an S172C (lanes 1 and 2) or S172A (lane 3) alteration were treated with (lanes 2 and 3) or without (lane 1) the cysteine-directed cross-linker SPDP. YfgL-His₆ and conjugated proteins were extracted under denaturing conditions and purified by nickel affinity spin columns. Purified protein complexes were separated on an SDS-PAGE gel and visualized by silver staining. (B) The presence of purified YfgL-His₆ (bottom) and YaeT (top) was confirmed by Western blot analysis using HisProbe-HRP and YaeT antibodies, respectively.