Fig 2.

YscF and LcrV can be immunoprecipitated (IP) after cross-linking. (A and B) A WT, ΔyscF (ΔF), or ΔlcrV (ΔV) Y. pseudotuberculosis strain was exposed to DTSSP and then lysed. Lysates were split and immunoprecipitated with LcrV or YscF antibodies. Cross-links within the immunoprecipitates were cleaved with BME. Proteins were separated by SDS-PAGE and analyzed by Western blotting with anti-LcrV (A) or anti-YscF (B). (C) LcrV was immunoprecipitated from lysates of Y. pseudotuberculosis exposed to sulfo-EGS with anti-LcrV antibody. Proteins were eluted from protein A beads by boiling in sample buffer. Proteins were analyzed by Western blotting with anti-LcrV (i), anti-YopD (ii), and anti-YopB (iii) antibodies. Each experiment was repeated twice, and representative blots are shown. Asterisks indicate incompletely stripped LcrV. Δ5N, ΔyopHEMOJN; Δ5NV, ΔyopHEMOJN ΔlcrV; Δ5NF, ΔyopHEMOJN ΔyscF; α, anti.