Western blotting was used to measure dCas9 expression in the TetR-DOZI-based inducible dCas9-GCN5 or -Sir2a system. Ring-stage parasites expressing both TetR-DOZI and the dCas9-fusion proteins were equally divided into two cultures and were treated with aTc at 0.5 μM or ethanol (vehicle control) for 30 h. Then, parasites were harvested for Western blotting with rabbit anti-FLAG antibodies (1:1,000, Abcam) and rabbit anti-PfAldolase as the primary antibodies and HRP-conjugated goat anti-rabbit IgG as secondary antibodies. The wild-type 3D7 was used as a negative control.
IFA was performed to detect the expression of dCas9 according to an established method (68 (link), 69 (link)). Briefly, parasitized RBCs were fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde, followed by quenching with 50 mM glycine. Fixed cells were then treated with 0.5% Triton X-100 and blocked in 3% bovine serum albumin (BSA) for 1 h. The anti-FLAG antibodies and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Sigma) were used as primary and secondary antibodies. Images were captured using a Nikon Eclipse E600 epifluorescence microscope.
IFA was performed to detect the expression of dCas9 according to an established method (68 (link), 69 (link)). Briefly, parasitized RBCs were fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde, followed by quenching with 50 mM glycine. Fixed cells were then treated with 0.5% Triton X-100 and blocked in 3% bovine serum albumin (BSA) for 1 h. The anti-FLAG antibodies and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Sigma) were used as primary and secondary antibodies. Images were captured using a Nikon Eclipse E600 epifluorescence microscope.
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