Divergent roles for the RH5 complex components, CyRPA and RIPR in human-infective malaria parasites
Fig 3
HA-tagging, localization and complex formation of PkRIPR.
(A) Schematic of single homologous recombination to generate C-terminally tagged parasite line, PkRIPR-HA. (B) Integration PCRs using genomic DNA from PkA1-H.1 (PK wt) parasites or PkRIPR-HA parasite clones I1 and G4. Using the primer pair PkRIPRextF1/PkRIPRutrRev, a 1595 bp fragment was amplified from wild type parasites, whereas primer pair PkRIPRextF1/HArev amplified a 1526 bp fragment only from transgenic PkRIPR-HA parasites. Positions of primer pairs are indicated in schematic (A) and size standards (kb) on the left of the gel (B). Primers used were PkRIPRextF1 (1), PkRIPRutrRev (2), HArev (3). (C) Southern blot of wildtype and transgenic parasite DNA digested with EcoRV/AvaI. Endogenous locus band, integration and episomal bands are indicated with arrows. (D) Immunoblot of purified schizont material solubilized in Laemmli sample buffer probed with anti-BiP and anti-HA (3F10) antibodies. Molecular mass standards are indicated on the left in kDa. (E) IFAs of schizonts and merozoites with micronemes not secreted (2nd row) or secreted (3rd row) were probed with anti-HA antibody (green), anti-PkAMA1 antibody, or anti-PkRhopH2 antibody (both red) and DAPI (Blue). The third panel shows an overlay of DAPI marking the nucleus with anti-HA and anti-PkAMA1; the fourth panel includes a differential interference contrast (DIC) image of the whole parasite. Scale bar = 2 μm. (F) Table of proteins interacting with PkRIPR-HA as identified by LC-MS/MS following immunoprecipitation. The three top-scoring, consistently identified proteins are listed. The number of peptides of three technical replicates of immunoprecipitates from PkRIPR-HA clone G4 or wild type parasites is displayed, as is the total peptide coverage, gene IDs and annotations (PlasmoDB release 25).