Fluorchem r system

Immunoblot analysis was performed as described previously (Wang et al. 2015 (link)). Briefly, 1 mL of culture was collected and resuspended in lysis buffer (20 mM Tris at pH 7.0, 10 mM MgCl₂, 1 mM EDTA, 1 mg/mL lysozyme, 10 µg/mL DNase I, 100 µg/mL RNase A, 1 mM PMSF, 1 µg/mL leupeptin, 1 µg/mL pepstatin) to a final OD₆₀₀ of 10 for equivalent loading. The cells were incubated for 10 min at 37°C, followed by addition of an equal volume of sodium dodecyl sulfate (SDS) sample buffer (0.25 M Tris at pH 6.8, 4% SDS, 20% glycerol, 10 mM EDTA) containing 10% 2-mercaptoethanol. Samples were heated for 15 min at 65°C prior to loading. Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, electroblotted onto immobilon-P membranes (Millipore), and blocked in 5% nonfat milk in phosphate-buffered saline (PBS) with 0.5% Tween-20. The blocked membranes were probed with anti-LytE (1:10,000) (Dobihal et al. 2019 (link)), anti-SigA (1:10,000) (Fujita and Sadaie 1998 (link)), anti-His (1:4000; Sigma), and anti-GFP (1:10,000) (Rudner and Losick 2002 (link)) antibodies diluted into 3% BSA in PBS with 0.05% Tween-20. Primary antibodies were detected using horseradish peroxidase-conjugated goat antirabbit or antimouse IgG (Bio-Rad) and the Super Signal chemiluminescence reagent as described by the manufacturer (Pierce). Signal was detected using a Bio-Techne FluorChem R system.