Figure 3. Antigen binding recognition by translocated sdAbs.

(A) Antigen expression (GFP) increases the level of T3sVgfp detected in the cytoplasm of HeLa cells. Western blot of “eukaryotic cytoplasm” (top panels) and “SLO-insoluble” (bottom panels) protein extracts from infected HeLa cell cultures transfected with the indicated plasmids: pEGFPN1 (GFP) or control pCS2+MT (mock). Infections were carried out for 150 min with EPEC wt and ΔescN strains harboring pT3sVgfp. The level of T3sVgfp in the cytoplasmic extracts of HeLa cells was developed with anti-E-tag mAb (T3sV_HH top panel). Absence of bacterial contamination in eukaryotic cytoplasm protein extracts was controlled with anti-DnaK mAb (DnaK top panel). Transfection and expression of GFP was controlled with and anti-GFP mAb (GFP panel). Levels of human cytoplasmic GAPDH were determined with anti-GAPDH mAb to control equal SLO pore formation in all samples and as a loading control. SLO-insoluble protein extracts (bottom panels) were developed with anti-E-tag mAb to show the level of T3sVgfp in bacteria and with anti-DnaK mAb to control bacterial attachment. (B) Immunoprecipitation of T3sVgfp:GFP complexes from the cytoplasm of infected HeLa cells. Western blot of input eukaryotic cytoplasm protein extracts and immunoprecipitated proteins with anti-E-tag mAb bound to protein G-Sepharose resin. Input extracts were obtained by SLO treatment from pEGFP-N1-transfected HeLa cell cultures infected with wt EPEC strain carrying pT3sVgfp or pT3sVamy. Purified T3sVamy was added to one aliquot of input extract from cells infected with EPEC/pT3sVamy (lane 3) to reach a level similar to the translocated T3sVgfp (lane 1). Input extracts were developed with anti-E-tag, anti-GFP, and anti-GAPDH mAbs (top panels). Immunoprecipitated (IP) proteins were developed with anti-E-tag and anti-GFP mAbs (bottom panels).