Translocation of C. trachomatis proteins into the cytoplasm of HeLa cells by Y. enterocolitica . HeLa cells were left uninfected (UI) or infected with Y. enterocolitica ΔHOPEMT strains expressing the indicated HA-tagged proteins. After 3 h of infection, extracellular bacteria were killed by the addition of gentamicin and the infected cells were incubated for additional 2 h. The infected cells were then fractionated into Triton-soluble and Triton-insoluble cell lysates that were subsequently analyzed by immunoblotting using anti-HA, anti-SycO and anti-tubulin antibodies, as indicated. Presence of HA-tagged proteins in the Triton-soluble cell lysates is indicative of translocation into the cytoplasm of HeLa cells. SycO is a strictly cytosolic Yersinia T3S chaperone [44, 51] and its immunodetection ensured that the presence of HA-tagged proteins in the Triton-soluble cell lysates was not a result of bacterial lysis during the fractionation. Additionally, the incapacity to detect HA-tagged RplJ (a C. trachomatis ribosomal protein) in the Triton-soluble cell lysates further indicated that this fraction did not contain bacteria or non-translocated bacterial proteins. Tubulin served as a loading control of the Triton-soluble cell lysates. The images shown are representative of three independent experiments.