Whole-cell and Supernatant Protein Analysis

Bacterial whole-cell and supernatant samples were prepared as before (7 (link), 8 (link)). Briefly, cultures were grown to an optical density at 600 nm (OD₆₀₀) of 0.5, and the OD 0.5 cultures were pelleted by centrifugation and resuspended in 50 μl of Laemmli buffer for the whole-cell samples. Supernatant samples were obtained by trichloroacetic acid (TCA) precipitation of cell-free supernatants (7 (link), 8 (link)). Protein samples were analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, probed with polyclonal anti-CpaA (1:1,000) (8 (link)) and/or monoclonal anti-RNA polymerase (1:2,000; BioLegend). Western blots were probed with IRDye-conjugated secondary antibodies and visualized with an Odyssey CLx imagining system (Li-COR Biosciences, Lincoln, NE).