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. Author manuscript; available in PMC: 2020 Oct 23.
Published in final edited form as: Nat Microbiol. 2020 Apr 13;5(6):848–863. doi: 10.1038/s41564-020-0702-4

Figure 2. Conditional knockdown strategies for efficient excision and localisation of the FIKKs.

Figure 2

(a) Description of the FIKK cKO strategy using the SLI method (Birnbaum, 2017). Schematic shows the structure of the fikk locus after integration and after Cre-mediated excision. (b) CRISPR/Cas9-mediated introduction of LoxPints into the fikk9.1 and fikk9.7 loci. RAP-treatment induces excision of all FIKKs located on chromosome 9 and expression of the Neomycin resistance cassette. (c) Western blots confirming correct expression and excision of FIKK kinases upon RAP-treatment. MAHRP1 antibody (bottom panel) demonstrates equal loading. FIKK7.1 samples were obtained by HA immunoprecipitation. Arrows – FIKK band at expected size (shown in labels). At least 3 independent western blots were performed for each FIKK with similar results. (d) Subcellular localisation pattern of a selection of FIKK kinases using antibodies against the C-terminal HA-tag fused to each FIKK kinase, MAHRP1 (Maurer’s cleft marker), and KAHRP (knob marker). DAPI was used for nuclear staining. For FIKK5, a late schizont is shown on the left panel, and a burst schizont on the right to demonstrate the localisation of the kinase in merozoites. Scale bar - 5μm. IFAs were performed at least 3 times for each FIKK with similar results.