Fig. 2. VSP citrullination is probably mediated by the PAD activity of ADI. (A) Top panels: confocal direct IFA was performed on permeabilized cells, showing a cytoplasmic distribution of ADI-HA (green) by using FITC-labeled anti-HA mAb and its partial colocalization (yellow in merge) with VSP9B10 (red) underneath the plasma membrane of the transgenic trophozoite. Nuclei are stained with DAPI (blue). Bottom panels: the same result was obtained using Alexa-Fluor-488–anti-ADI (green) in wild-type cells. Texas-Red–9B10 mAb was used to visualize VSP9B10 (red). Scale bars: 10 µm. (B) Western blotting of G. lamblia homogenates expressing different VSPs that reacted with anti-citrulline pAb (left panel). The same filter membranes were stripped, cut and reacted with 5C1, 9B10 and G10/4 mAbs against VSP1267, VSP9B10 and VSPH7, respectively (right panel), indicating that these VSPs are citrullinated. STD, molecular weight standard. (C) Dot-blotting to detect citrullination of the H₆-CRGKA peptide after incubation with the purified recombinant ADI-HA (ADI-HA). A non-related purified enzyme ESCP-HA was used as a negative control. Dot-blotting to detect H₆-CRGKA was performed using anti-H₆ mAb. (D) Top panel: specific citrullination of the CRGKA tail is shown. Western blotting using anti-citrulline pAb performed after immunoprecipitation with anti-HA mAb of H7-transgenic trophozoites. Bottom panel: the presence of H7-HA, H7-tail and R-H7 after immunoprecipitation was analyzed using anti-HA mAb labeled with alkaline phosphatase. UT, untransfected cells.