Western Blot Detection of PilA-FLAG Proteins

To verify the production of PilA-FLAG proteins, cell lysates were prepared as described previously with minor modifications (77 (link)). Briefly, after overnight growth, the bacterial culture was back diluted 1:100 in 2 ml of LB and grown at 37°C for the indicated amount of time (for time course experiments) or up to an OD₆₀₀ of ∼0.65. At the time of harvesting, the bacteria were centrifuged for 3 min, and the pelleted cells were resuspended in 2× Laemmli buffer, whereby the volume was adjusted according to the total number of bacteria (100 μl buffer per OD₆₀₀ unit). The resuspended samples were incubated at 95°C for 15 min. Proteins were separated by SDS-PAGE using 15% resolving gels and blotted onto\polyvinylidene difluoride (PVDF) membranes as previously described (76 (link)). Primary monoclonal antibodies against the FLAG tag (ANTI-FLAG M2; Sigma-Aldrich) were used at 1:2,000 dilution, and goat anti-mouse antibody—horseradish peroxidase (HRP) served as the secondary antibody (diluted 1:5,000; Sigma-Aldrich). Sigma70 was detected as a loading control using Direct-Blot anti-E. coli Sigma70-HRP-conjugated antibodies at a dilution of 1:10,000 (BioLegend, USA distributed via Brunschwig, Switzerland). Lumi-Light^PLUS Western blotting substrate (Roche) served as the HRP substrate. Luminescent signals were detected using a ChemiDoc XRS+ station (Bio-Rad).