Protein expression and analyses

SDS-polyacrylamide gels and quantitative immunoblotting analyses were used to analyze the protein profiles and expression levels of mutated AdeB proteins in AbΔ3 and AbΔ3-Pore strains. Membrane fractions were isolated after 5 h of induction from A. baumannii cells by ultracentrifugation as described before (32). Proteins were normalized to the same total protein concentration, analyzed by 8% SDS-PAGE, and transferred to PVDF membranes for immunoblotting with primary polyclonal antiAdeB antibodies (ThermoFisher Scientific) and a secondary alkaline phosphatase-conjugated antirabbit immunoglobulin antibody (Sigma). The 5-bromo4-chloro-3-indolylphosphate and nitroblue tetrazolium substrates were used to visualize the bands.