Figure 1

LI0666 is a substrate of the Yersinia type 3 secretion system. A Schematic diagram of LI0666. The EPIYA motif is highlighted in yellow. B T3SS-competent Y. enterocolitica MRS40_∆YopHOPEM expressing HA-tagged proteins (LI0666, LI0666_N40-YopE_∆N15, RplJ, YopE) and T3SS-null Y. enterocolitica MRS40_∆yscF expressing HA-tagged proteins (LI0666, LI0666_N40-YopE_∆N15, YopE) were cultivated in BHI under T3SS-repressive (+ Ca²⁺), or -inductive (−Ca²⁺) conditions. Bacteria were grown for 4 h at 37 °C in the presence of 0.2% L-arabinose to induce T3SS and transgene expression. Equal amounts of Y. enterocolitica MRS40_∆YopHOPEM cultures expressing HA-LI0666 and HA-RplJ, Y. enterocolitica MRS40_∆yscF cultures expressing HA-LI0666 and HA-YopE, and Y. enterocolitica MRS40_∆YopHOPEM cultures expressing HA-YopE were centrifuged to separate the cell-free culture supernatants (CS) from the whole-cell pellets (WC), and material corresponding to 0.10 OD₆₂₀ per mL of the original cultures for CS and 0.02 OD₆₂₀ per mL for WC was resolved on 12% (w/v) polyacrylamide gels. Specific proteins were detected by immunoblot with anti-HA followed by visualization with alkaline phosphatase–conjugated secondary antibodies and development with ECL.