YopE extracted with digitonin from HeLa cell monolayers infected with Yersinia defective in YopB is degraded in a protease protection assay. HeLa cell monolayers, infected for 3 h with wild-type Y. enterocolitica (wt) or a yopB1 mutant were either treated (+) or not treated (−) with PK. Phenylmethylsulfonyl fluoride was added to all cultures to block protease activity. Infected cultures were then extracted with either digitonin or SDS. Resulting lysates were centrifuged, and soluble proteins were separated on an SDS–12% polyacrylamide gel and subjected to Western blot analysis with antibodies against YopE (A) or with antibodies against SycE (B). ∗, results correspond to data in reference 12.

SDS extraction of bacteria solubilizes both YopE and the intrabacterial chaperone SycE. Wild-type Y. enterocolitica or a yopB1 mutant was grown in tissue culture dishes without HeLa cells for 4 h. Collected bacteria were centrifuged and resuspended in 500 μl of PBS containing either 500 μl of digitonin (1%) or SDS (1%). Bacteria were incubated at room temperature for 20 min. Cleared lysates were fractionated by SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis with antibodies against YopE (A) or SycE (B).