Fig. 7. Degradation of anti-VSG IgG. BSF wild-type, mca2/3mca5 and triple RNAi lines, with or without induction with tetracycline for 8 hours. Parasites were labelled with anti-VSG221 IgG at 4°C in HMI-9 prior to incubation at 37°C for 5 and 10 minutes in HMI-9. (A) Parasites were fixed and stained with anti-rabbit-FITC conjugate (green) and DAPI (blue). Insets, DIC images of the parasite. Bars, 10 µm. (B) Whole-cell lysates were prepared after the 30-minute incubation and 5x10⁶ cell equivalents subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with an HRP-conjugated anti-rabbit IgG. Lane 1, wild-type BSF; lane 2, mca2/3mca5. EF-1 served as a loading control. (C) TCA precipitations of the culture medium were resuspended, subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with an HRP-conjugated anti-rabbit IgG. Lane 1, without anti-VSG221 IgG; lane 2, with anti-VSG221 IgG in the absence of parasites; lane 3, wild-type BSF; lane 4, mca2/3mca5 BSF. Anti-VSG221 IgGs are indicated together with degradation products.