SDS-PAGE and western blot analysis

Secreted protein extracts and whole cell extracts were separated on a 12.5% SDS-PAGE gel. To detect total protein, Coomassie staining was performed. When specific proteins were detected, proteins were electrotransferred onto a PVDF membrane (Bio Rad). For immunodetection the following antisera were used; monoclonal anti-FLAG (Sigma) polyclonal anti-SopE [56] and monoclonal anti-GreA (Neoclone). Detection was with a HRP-conjugated secondary antibodies and ECL Prime Western Blotting detection reagent (GE Healthcare). Visualization of the detected bands was performed using Molecular Imager ChemiDoc XRS System and Quantity One software (Bio Rad). Prior to western blot analysis from whole cell extracts, the protein content of samples normalized by OD_600nm (biomass of original cultures) was corroborated by Coomassie staining. Equally, loading of secreted protein extract was normalized to the culture biomass (OD_600nm). Routinely, normalization by biomass of cultures grown to obtain secreted protein extracts was corroborated by SDS-PAGE and Coomassie staining of the cell extract from the cultures. Moreover, immunodetection of the cytoplasmic cyclic AMP receptor protein CRP was performed in secreted protein extracts to exclude contamination with cellular proteins. A representative control experiment performed on a secreted protein extract is shown in S8 Fig.