Quantifying Bacterial Secretion Systems

Overnight cultures were back-diluted in fresh LB medium to an OD₆₀₀ of 0.025 and grown at 37°C with shaking until they reached an OD₆₀₀ of 0.35 to 0.5 (AbCAN2) or 0.4 to 0.7 (Ab17978). The cells were then pelleted by centrifugation. The cells were resuspended in Laemmli buffer to a final OD₆₀₀ of 10, while the supernatant fraction was centrifuged once again (as above) to pellet residual cells. Supernatant proteins were subsequently precipitated with trichloroacetic acid, as previously described (39 (link)). Optical desity-normalized volumes of whole cells or supernatants were loaded onto 15% (for Hcp) or 8% (for VgrGs) SDS-PAGE gels for separation, transferred to a nitrocellulose membrane, and probed with polyclonal rabbit anti-Hcp (1:1,000) (39 (link)), polyclonal rabbit anti-6×His (1:2,000; Invitrogen, Waltham, MA) or monoclonal mouse anti-RNA polymerase (1:2,600; BioLegend, San Diego, CA). Western blots were then probed with IRDye-conjugated anti-mouse and anti-rabbit secondary antibodies (both at 1:15,000; LI-COR Biosciences, Lincoln, NE) and visualized with an Odyssey CLx imaging system (LI-COR Biosciences).