Fig. 5
From: Functional characterisation of components in two Plasmodium falciparum Cullin-RING-Ligase complexes
Identification of MCM complex in P. falciparum. (a) Immunoblots showing C-terminal tagging of PfMCM4 in DiCre transgenic strain parasites by anti-Flag antibodies. (b) Volcano plot illustrating the log2 protein ratios in anti-FLAG immunoprecipitations from parasite lysate comparing DiCre (red) and PfMCM4-Flag (blue). Proteins were deemed differentially regulated if the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. Proteins of interest to this study are further labelled with protein names. (c) Immunoblot of MCM4-Flag containing DiCre, PfSKP1-HA and PfRBX1-HA schizont stage parasites treated with DMSO or rapamycin. Anti-Flag, Aldolase and HA antibodies were used to identify levels of PfMCM4, loading and PfSKP1/PfRBX1 protein levels respectively. Densitometry of n = 2 western blots with mean ± s.d are plotted next to immunoblot. (d) Immunoblot of anti-Flag immunoprecipitation of MCM4-Flag from DiCre-PfSKP1-HA + MCM4-Flag transgenic schizont stage parasites pre-treated with DMSO or 10 nM Rapamycin. Input (I), Unbound (U) and Eluate (E) fractions were analysed using antibodies against MCM4 (anti-Flag), Ubiquitin (anti-ubiquitin) and PfSKP1 (anti-HA). Full-length and truncated PfSKP1-HA is denoted by one and two blue circles respectively. Note that the anti-HA panel has been overexposed so that the SKP1 HA-Neon Green fusion can be observed. (e) Immunoblot of anti-Flag immunoprecipitation of MCM4-Flag from DiCre-PfRBX1-HA + MCM4-Flag transgenic schizont stage parasites pre-treated with DMSO or 10nM Rapamycin. Input (I), Unbound (U) and Eluate (E) fractions were analysed using antibodies against MCM4 (anti-Flag), Ubiquitin (anti-ubiquitin) and PfRBX1 (anti-HA). Full-length and truncated PfRBX1-HA is denoted by one and two red circles respectively. (f) Immunofluorescence analysis of MCM4-Flag tagged DiCre-PfSKP1-HA strain schizont stage parasites. Parasites were treated with DMSO or Rapamycin to induce PfSKP1 deletion and PfMCM4, parasite membrane integrity and DNA levels were visualized with anti-Flag, anti-PfGAP45 antibodies and DAPI staining respectively. (g) Immunofluorescence analysis of MCM4-Flag tagged DiCre-PfRBX1-HA strain schizont stage parasites. Parasites were treated with DMSO or Rapamycin to induce PfRBX1 deletion and PfMCM4, parasite membrane integrity and DNA levels were visualized with anti-Flag, anti-PfGAP45 and DAPI respectively.
