Figure 5

Secretion of YopE_1–52-DHFR and YopE_1–53-DHFR after inhibition of protein biosynthesis. Yersiniae (Y. enterocolitica strain WA-314, abbrev. WT, and the secretion deficient yscV mutant) expressing YopE_1–52-DHFR and YopE_1–53-DHFR, respectively, were cultured for 2 h at 37°C in BHI supplemented with CaCl₂. Then 50 μg/ml of tetracycline was added to inhibit protein synthesis and cultures were incubated for another 20 min. Then bacteria were pelleted and resuspended in BHI depleted of Ca²⁺ to induce Yop secretion in the presence of tetracycline. After 20 min of continued incubation bacteria were pelleted and whole cell lysates (P) and TCA-precipitated supernatants (S) were subjected to SDS-PAGE; ten times more supernatant than cell pellet was loaded. Subsequently, immunoblotting was performed with monoclonal anti-DHFR antibodies.