Mouse anti rnap

We pelleted cells from each condition at 5,000 g for 10 min. After the supernatant was decanted, the pellets were resuspended in 60 μl of B‐PER (Thermo Scientific) containing complete Mini ethylenediaminetetraacetic‐acid‐free protease inhibitor cocktail (Roche), and cells were lysed for 30 min at room temperature. After pelleting the cell debris by centrifugation at 15,000 g for 5 min, we transferred 40 μl of clear lysate to a new tube. We determined protein concentrations using a Bradford assay reagent (Thermo Scientific) and denatured proteins by adding 10% sodium dodecyl sulfate [SDS] (Sigma‐Aldrich) to a final concentration of 2%. Next, 60 μg of total protein from each cell lysate were loaded and separated by 12% SDS‐polyacrylamide gel electrophoresis. The proteins were transferred onto a polyvinylidene fluoride membrane (Immobilon, 0.45 μm, Millipore) and probed via immunoblotting. We used the following antibodies for immunoblotting: rabbit anti‐TcpA (Taylor et al, 2004 (link); 1:2,500), mouse anti‐RnaP (Biolegend; 1:2,500), anti‐rabbit horseradish peroxidase‐conjugated (Promega, 1:2,500), and anti‐mouse horseradish peroxidase‐conjugated (Invitrogen; 1:2,500). The immunoblots were developed with the SuperSignal West Pico chemiluminescent kit (Pierce).