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Fig. 4. Post-translational modifications of ADI. (A) Western blotting using anti-HA mAb shows ADI-HA in ADI-transgenic trophozoites. Multiple bands are also obtained using specific anti-ADI pAb in wild-type cells. The band of 64-66 kDa corresponding to the predicted protein sequences is observed in both cases (arrowhead), together with degradation products (gray arrows) including the low-weight band (gray arrow with asterisk) found in the peptide pull-down (see Fig. 1). Also, for both HA-tagged and native ADI, an increase in molecular mass from a 64-66 kDa to a 85 kDa band is shown (black arrow), suggesting that ADI undergoes post-translational modification. STD, molecular weight standard. (B) Western blot assays using anti-SUMO1 mAb recognize an 85 kDa band (arrow) in both WB/1267 (WB) and GS/H7 (GS) G. lamblia clones. The same filter membrane was stripped and re-blotted with anti-ADI pAb, showing a perfect match with the higher band that is recognized by the anti-SUMO1 mAb. To confirm the lack of residual primary antibodies after stripping, only the secondary antibody was added to the stripped blots, showing no signal (Control). (C) Western blotting using biotin-conjugated anti-ADI pAb was performed to detect ADI bands after immunoprecipitation with anti-SUMO1 mAb. (a) ADI in lysate before IPP; (b) ADI after immunoprecipitation by using 0.1 µg of anti-SUMO1 mAb (arrow); (c) ADI after immunoprecipitation by using 1 µg of anti-SUMO1 mAb (arrow); (d) control using a non-related anti-HA mAb; and (e) supernatant of sample c.

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