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	FIGURE 1. Sequence analysis of PfRCC-PIP. (A) Schematic representation of PfRCC-PIP. The RCC and PIP regions with the RVXF motif (in black), two RCC1 (in blue), and one RCC1_2 (in red) motifs are indicated. The portion of PfRCC-PIP detected by Y2H is highlighted in gray. (B) Alignments of the two RCC1 motifs of PfRCC-PIP with RCC1 consensus motif (pfam00415) and RCC1_2 of PfRCC-PIP with RCC1 signature 2 consensus motif (Prosite PS00626). (C) Predicted model of the portion of PfRCC-PIP containing the RCC1 motifs (AA 140–424), based on the structure of human RCC1 (D) (BAA00469.1) (see text footnote 1). Beta sheets are represented by arrows, RCC1 motifs are colored in blue and RCC1_2 motif in red.
	FIGURE 2. Targeted gene disruption of the PfRCC-PIP locus in P. falciparum. (A) Disruption of PfRCC-PIP by a knock-out strategy involving single homologous recombination using the pCAM-BSD vector. The pCAM-BSD construct, the blasticidin-resistance cassette (BSD), the location of the primers used for PCR analysis and the locus resulting from integration are shown. (B) Diagnostic PCR analysis of pCAM-BSD-PfRCC-PIP-transfected 3D7 cultures; lanes 1 to 3 correspond to DNA extracted from wild type 3D7 parasites and lanes 4 to 6 to DNA extracted from transfected parasites. Lanes 1 and 4 represent the detection of the wild type locus (PCR with p51-p52); lanes 2 (presence of non-specific band) and 5 (presence of the specific band at the expected size) represent the detection of the construct (PCR with p53-p54) and lanes 3 and 6 correspond to the integration at the 5′ end of the insert (PCR with p55-p54).
	FIGURE 3. Direct interaction between the PIP region of PfRCC-PIP and PfPP1 and involvement of the RVXF motif. (A) Interaction in a yeast two-hybrid assay between the PIP region of PfRCC-PIP wild type (WT) and mutated (980KSASA984), with PfPP1 wild type and mutated (255FF256/AA). Constructs in pGBKT7 were inserted into mat α yeast (Y187 strain), pGADT7 empty and pGADT7-RCC-PIP were inserted in mat a yeast (Y2HGold strain). After transformation, mating experiments were carried out. Diploids were checked on Ddo medium, interactions and strong interactions were examined on Tdo and Qdo medium, respectively. The expression of proteins was checked by western blot analysis of extracts prepared from each yeast strain and revealed with anti-cMyc mAb for wild type and mutated (255FF256/AA) PfPP1 (B) and anti-HA mAb for wild type (C) and mutated (D) PIP region of PfRCC-PIP. (E) Interaction between PfPP1 and PIP region of PfRCC-PIP analyzed by immunoprecipitation. Beads coupled to antibodies raised against PfRCC-PIP or to control antibodies from pre-immune serum were incubated with wild type or mutated PIP region of RCC-PIP and then with PfPP1-GST. Beads were washed, suspended in Laemmli buffer, and eluates were separated by SDS–PAGE and transferred onto nitrocellulose membrane. Immunoblot analysis was performed with the anti-PfRCC-PIP antiserum (upper panel) and with anti-GST antibody (lower panel) to detect PfPP1. Lane 2 shows the interaction of PfPP1 with the wild type PIP region of PfRCC-PIP and lane 4 shows the absence of interaction with the 980KSASA984-mutated protein. As controls, an input of PfPP1-GST (lane 5), the wild type PIP region of PfRCC-PIP (lane 6) or 980KSASA984-mutated (lane 7) were used.
	FIGURE 4. Tagging of endogenous RCC-PIP in P. falciparum and localization studies. (A) Knock-in strategy using the vector pCAM-BSD-HA that allows the insertion of an HA epitope at the C-terminus of PfRCC-PIP with a single homologous recombination. The pCAM-BSD construct, the blasticidin-resistance cassette (BSD), the location of the primers used for PCR analysis and the locus resulting from integration are shown. (B) Diagnostic PCR analysis of pCAM-BSD-HA-PfRCC-PIP-transfected Pf3D7 cultures; lanes 1 to 3 correspond to DNA extracted from wild type Pf3D7 parasites and lanes 4 to 6 to DNA extracted from transfected parasites. Lanes 1 and 4 represent the detection of the wild type locus (PCR with p25-p26); lanes 2 and 5 represent the detection of the construct (PCR with p27-p28); and lanes 3 and 6 correspond to the integration at the 5′ end of the insert (PCR with p29-p28). The presence of a PCR product in lane 6 (arrow) confirms the integration of an HA-tagged PfRCC-PIP gene in the locus. (C) Cellular distribution of PfRCC-PIP-HA analyzed by immunofluorescence with anti-HA antibodies. The PfRCC-PIP protein appears in green and the nucleus is stained in blue by DAPI. Upper and lower panels show one erythrocyte infected by one trophozoite and 3 trophozoites, respectively. The fluorescent signal is detected at the periphery of the nucleus. (D) PCR on reverse transcribed RNA from wild type (lanes 1 and 2) or knock-in PfRCC-PIP-HA parasites (lanes 5 and 6). Non-reverse transcribed RNA from PfRCC-PIP-HA parasites was used as negative control (lanes 3 and 4). PCR were performed with primers p29 and p28 to detect the integration (lanes 2, 4, and 6), and p25 and p26 for the control (lanes 1, 3, and 5). The absence of any band in lane 3 indicates the non-contamination of RNA by genomic DNA, and the presence of a fragment at the expected size in lane 6 (arrow) confirms the presence of HA-tagged PfRCC-PIP transcripts in PfRCC-PIP-HA parasites.
	FIGURE 5. Expression of PbPP1-HA in P. berghei. (A) The expression of PbPP1-HA (lane 2) was checked in transfected P. berghei parasites by western blot analysis with an anti-HA antibody, and parental parasites were used as control (lane 1). (B) The solubility of PbPP1-HA in transfected parasites was assessed by immunoprecipitation from the soluble fraction of these parasites. Lanes 1 and 2, correspond to insoluble and soluble fractions of PbPP1-HA parasites, respectively. Lanes 3 and 4 correspond to the immunoprecipitation of the soluble fraction from parental and PbPP1-HA parasites, respectively, using anti-HA beads. The membrane was probed with anti-HA antibodies.
	FIGURE 6. Expression of the PIP region of PfRCC-PIP in Xenopus laevis oocytes and role on the induction of Germinal Vesicle BreakDown. (A) Production of the PIP region of PfRCC-PIP in Xenopus oocytes. 15 h after micro-injection of the PIP region of PfRCC-PIP mRNA in Xenopus oocytes, the production of the corresponding protein was checked by western blot probed with an anti-HA antibody. A native oocyte lysate was used as negative control in lane PG. (B–D) Role of the PIP region of PfRCC-PIP on the induction of Germinal Vesicle BreakDown (GVBD). Xenopus oocytes were micro-injected with mRNA of PIP region PfRCC-PIP and were then incubated with or without progesterone (PG) at different concentrations, and GVBD was observed 16 h post injection (B,C) or every 3 h post injection (D). (B) Percentage of GVBD induced by PG (10 μM), by the PIP region of PfRCC-PIP, with or without PG incubation. (C) Percentage of GVBD induced by the PIP region of RCC-PIP in presence of increasing concentrations of PG (0.1 nM to 10 μM). In parallel, oocytes without micro-injection of the PIP region of RCC-PIP were used as control and incubated in the same concentrations of PG. Results are expressed as GVBD percentage (+/–SEM, n = 2), and the Mann-Whitney test was used to evaluate the significance with control oocytes (PG only). ∗P < 0.05. (D) Kinetic analysis of GVBD induced by wild type vs. mutated PIP region of RCC-PIP in presence of 0.1 nM of PG. (E) Kinetic analysis of protein expression in oocytes lysates after microinjection of mRNA encoding wild type vs. mutated PIP region of PfRCC-PIP. The membrane was probed with anti-HA mAb. (F) Interaction between XePP1 and the PIP region of PfRCC-PIP analyzed by co-immunoprecipitation. The XePP1-PfRCC-PIP complex was immunoprecipitated (IP) with anti-XePP1 antibodies from microinjected Xenopus oocytes, separated by SDS–PAGE and transferred to a nitrocellulose membrane. Western blot (WB) analysis was performed with anti-XePP1 antibodies (lower panel) or anti-HA antibodies (recognizing PfRCC-PIP) (upper panel). A band is observed in upper panel in lysates from oocytes microinjected with wild type PIP region of PfRCC-PIP 3 h after microinjection, indicating an interaction with XePP1.
	FIGURE 7. Interaction between RCC region of RCC-PIP and PfCDPK7. (A) Interaction between the RCC region of RCC-PIP and PfCDPK7 in yeast two-hybrid approach. The RCC region of PfRCC-PIP (AA 1–399) was cloned in pGBKT7 vector and the construct was inserted in mat α yeast (Y187). pGADT7 transformed with a fragment of PfCDPK7 (AA 994–1291) isolated from Y2H screening as a potential partner of PfRCC-PIP, was inserted in mat a (Y2HGold) yeast. After mating, diploids were checked on Ddo medium, interactions and strong interactions were examined on Tdo and Qdo medium, respectively. pGADT7 empty, pGBKT7 empty and pGBKT7-laminin were inserted in corresponding yeast strains and used as controls. (B) Direct interaction between RCC region of PfRCC-PIP and PfCDPK7 (AA 994–1291) assessed by GST pulldown assay. Glutathione beads alone (lane 1) or coupled with GST (lane 2), or with PfCDPK7-GST (lane 3) were incubated with the His-tagged recombinant RCC region of PfRCC-PIP. After washes, proteins bound to the beads were separated by SDS–PAGE and blotted onto nitrocellulose. Immunoblot analysis was performed with anti-GST mAb (upper panel) recognizing GST and PfCDPK7-GST proteins and anti-His mAb antibodies (lower panel) recognizing the recombinant RCC region of PfRCC-PIP. As a control, an input of His-tagged RCC region of PfRCC-PIP was used (lane 4).

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