Figure 1

Protease protection assay on cytosolic YopE_1–53-DHFR prepared from spheroplasts. Spheroplasts were prepared from yersiniae induced for Yop expression and treated with 1 mM methotrexate (MTX), 1 mM aminopterin (APT) or untreated, respectively. Spheroplasts were washed three times and subsequently lysed by osmotic shock, then soluble supernatants after centrifugation (cytosolic fractions) were treated with thermolysin (0, 1, 10 μg/ml). As "control", cytosol from untreated yersiniae was incubated with 1 mM MTX and 1 mM APT, respectively, prior to thermolysin exposition. After incubation at 20°C for 15 minutes samples were loaded on an SDS gel that was electroblotted. Immunostaining was performed with anti-DHFR monoclonal antibodies.