Immunoblot Analysis of InlB Expression

The restoration of InlB expression was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). Membrane-bound protein samples were prepared from overnight L. monocytogenes cultures grown in BHI broth supplemented with 0.2% charcoal to activate the PrfA regulon as described previously (Ermolaeva et al., 2004 (link)). SDS–PAGE was performed in accordance with the generally accepted techniques. Cell lysates of L. monocytogenes were boiled for 10 min, separated on 10% SDS–PAGE and transferred onto PVDF membrane. InlB was visualized with polyclonal primary anti-InlB antibodies were obtained as described earlier (Povolyaeva et al., 2020 (link)) and donkey anti-rabbit IgG HRP-labeled antibodies (Abcam, London, United Kingdom).