0.45 μm pore size nitrocellulose membrane

The cells were lysed in lysis buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40, a protease and phosphatase inhibitor mixture (Roche)] and centrifuged for 15 min 20,000×g at 4 °C to obtain the cell lysates. Next, the cell lysate concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). The total protein sample was prepared using 5× sodium dodecyl sulfate (SDS) sample buffer and heated at 99 °C for 10 min. Proteins were separated on a 10% SDS–polyacrylamide electrophoresis gel and transferred to a 0.45-μm pore size nitrocellulose membrane (GE Healthcare Life Science). The membrane was incubated overnight at 4 °C with primary antibodies in TBS-T [150 mM NaCl, 20 mM Tris–HCl (pH 8.0), and 0.05% Tween-20] containing 3% BSA, followed by secondary antibody incubation using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Fab) (Enzo Life Sciences) in 5% skim milk dissolved in TBS-T at 26 °C for 2 h. Proteins were visualized with an ECL western blotting reagent (BioNote) and analyzed on a Fusion Solo-S image analyzer (Vilber). Protein band intensities were quantified and analyzed using ImageJ software.

Overnight cultures from a single colony were diluted 1:100 into T medium with or without NRES. Cultures were grown to mid-log phase (OD₆₅₀ of approximately 0.6), and samples containing an equivalent number of cells were resuspended in Laemmli SDS sample buffer (58 (link)). Samples were resolved by electrophoresis through 10% SDS-polyacrylamide gels, and the proteins were visualized by Coomassie brilliant blue staining or immunoblotting. On the Coomassie gels, the positions of the OmpT and OmpU proteins were determined by comparisons with previous studies of the wild-type strain performed with ompT and ompU mutant strains (59 (link)). The proteins for immunoblotting were transferred to a 0.45 μm-pore-size nitrocellulose membrane (GE Healthcare). ToxR was detected using rabbit polyclonal anti-ToxR antiserum (a gift from R. K. Taylor) (diluted 1:1,000). V5 fusion proteins were immunodetected by the use of mouse monoclonal anti-V5 antibodies (Sigma-Aldrich; catalog no. V8012) (diluted 1:10,000). To visualize the proteins, either horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (Bio-Rad Laboratories) (diluted 1:10,000) was used. To ensure equal levels of loading, the relative densities of the immunoblotted samples were assessed by Coomassie staining of a duplicate gel.

Inner and outer membranes were isolated by membrane fractionation as described above and resuspended in Laemmli SDS sample buffer (5% β-mercaptoethanol, 3% [wt/vol] SDS, 10% glycerol, 0.02% bromophenol blue, 63 mM Tris-Cl [pH 6.8]) (69 (link)), and boiled for 5 min. Samples were electrophoresed in quadruplicate (10%) SDS-polyacrylamide gels for separation. Proteins from three gels were transferred to a 0.45-μm-pore-size nitrocellulose membrane (GE Healthcare) and incubated with either rabbit polyclonal anti-IcsA antibody (Edwin Oaks, Walter Reed Army Institute of Research) diluted 1:10,000, rabbit polyclonal anti-SecA antibody (Donald Oliver, Wesleyan University) diluted 1:10,000, or rabbit polyclonal anti-OmpA antibody (Donald Oliver, Wesleyan University) diluted 1:5,000. Proteins were detected using horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:5,000). Signal was detected by developing the blot with Pierce ECL detection kit (Thermo Fisher). Proteins from the fourth gel were visualized by Coomassie brilliant blue staining and used to assess equal loading of samples for immunoblotting.