FIG. 4.

Tir delivery into HeLa cells under Ca²⁺-depleted conditions. HeLa cells were incubated for 15 or 30 min (C) or for 120 min (A and B) with bacterium-free supernatant of EPEC strain E2348/69 or DA-EPEC strain 3431 grown without Ca²⁺ (A and C) or recombinant His-tagged Tir (B). After repeated washing, the cells were fractionated as described in the text. Cytoplasmic (CF) and membrane (MF) fractions were separated by SDS-12.5% PAGE (A) or SDS-10% PAGE (B and C) and transferred to nitrocellulose membranes. The immunoblotting analysis was performed by using anti-Tir antibody.