Fig. 1. G. lamblia ADI is associated with VSPs through their cytoplasmic tail. (A) Left: representation of the pull-down assays. Right: SDS-PAGE and Coomassie staining show an 85 kDa and an 13 kDa protein from both GS/H7 (GS) and WB/1267 (WB) isolates. Identification of these bands as being ADI was performed by LC/MS-MS. Controls without peptide fail to pull down any protein. (B) Protein-protein interactions were detected by the ability of yeast cells (AH109) to grow on selective plates. In the upper-left panel, the expression of the entire VSPH7 protein (H7-BD), the entire VSP1267 protein (1267-BD) and VSP1267 lacking its cytoplasmic tail (1267-tail-BD) with ADI (ADI-AD) is revealed by the presence of white colonies in minimal medium lacking leucine and tryptophan (–L/–T medium). Interaction of ADI-AD with both VSPH7-BD and VSP1267-BD is shown in the bottom-left panel by the growth of yeast colonies in plates lacking tryptophan, leucine and histidine [TDO (triple-dropout medium) plates]. No interaction between ADI and 1267-tail-BD is observed. Controls of the methodology include ESCP-BD–MuA-AD interaction (+) and emptyBD–ADI-AD vector (–). (C) Schematic representation of wild-type VSPH7 and transgenic VSPH7 proteins. The VSPH7 ORF contains a signal peptide, an extracellular domain, a transmembrane domain and a cytoplasmic tail. H7-HA has a HA epitope sequence at the C-terminus. H7-tail possesses the HA epitope right after the transmembrane domain of H7. R-H7 is VSPH7 containing a point mutation of the R residue of its cytoplasmic tail. (D) H7-HA and R-H7, but not H7-tail, co-immunoprecipitate with ADI. Antibody against ADI was used to immunoprecipitate comparable amounts of protein from WB transgenic cells. Western blotting of original lysate was stained with anti-HA mAb labeled with alkaline phosphatase (Lysate, bottom panel). UT, untransfected cells; STD, molecular weight standard.