Vandomme A , Fréville A , Cailliau K , Kalamou H , Bodart JF , Khalife J , Pierrot C .

	Figure 1. Molecular cloning and sequence analysis of PfPTPA. (a) Analysis of PfPTPA (PF3D7_1430100) amino acid sequence. PfPTPA was aligned with the human PTPA (CAA60163.1) using ClustalW Multiple Alignment (BioEdit). The identical residues are highlighted in black and similar residues in grey. Stars symbolized amino acids involved in PTPA/PP2A interaction in human; (b) Phylogenic tree of the PTPA family. A maximum likelihood tree was generated from the 21 PTPA sequences using MEGA5 [45] under the JTT + G + I model with 100 bootstrap repetitions. Outgroups are formed by PP2A inhibitor 2 orthologs (I2PP2A, outgroup 1) PP1 inhibitor 2 orthologs (I2PP1, outgroup 2) and PP1 inhibitor 3 orthologs (I3PP1, outgroup 3); (c) Crystal structure of HuPTPA (PDB: 2IXM). Amino acid residues involved in PTPA/PP2A interaction are shown in red; (d) Structural model of PfPTPA based on the crystal structure of HuPTPA (PDB: 2IXM) using the ModBase server [46]. Amino acid residues studied in this work for the PTPA/PP2A interaction are shown in red.
	Figure 2. Expression of pfptpa and pfpp2a gene by Plasmodium falciparum and localization (a) Representation of pfptpa (black bars) and pfpp2a (grey bars) RNA expression during the erythrocytic life cycle of P. falciparum. Data plotted are from PlasmoDB [44]; (b) Schematic representation of the pARL2-pfptpa-gfp and pARL2-pfpp2a-gfp vectors used for episomal expression of both PfPTPA and PfPP2A; (c) The expression of both proteins was checked by western blotting with anti-GFP antibodies after separation on 15% SDS-PAGE. Lane 1 represents the extract of wild type parasites. Lane 2 and 3 represent extracts from PfPTPA and PfPP2A transfected parasites respectively. Expression and localization of PfPTPA-GFP (d) and PfPP2A-GFP (e) throughout the erythrocytic cell cycle of P. falciparum were analyzed by fluorescence microscopy after transfection as described under experimental section.
	Figure 3. Interaction studies of PfPTPA to PP2A. (a) Binding of PfPTPA with PfPP2A in Xenopus oocyte. Co-immunoprecipitation of the PfPTPA-PfPP2A complex with anti-His antibodies (recognizing recombinant PfPTPA tagged with 6-His) (lanes 2 and 3) from microinjected Xenopus extracts. The anti-mouse IgG antibody (lane 1) was used as a control. Immunoprecipitates from Xenopus oocytes microinjected with water (−) or PfPTPA (+) were eluted and separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblot analysis was performed with anti-His antibodies (recognizing PfPTPA) (upper panel) or anti-Myc antibodies (recognizing PfPP2A) (lower panel); (b) Interaction of PfPTPA with HuPP2A in vitro assessed by ELISA based assay. Increasing quantities of biotinylated recombinant PfPTPA were added to wells coated with human PP2A (100 ng/well) or BSA (100 ng/well) as a negative control. Results are means ± SEM of two independent experiments performed in duplicate (stars (*) represent significant differences p = 0.01); (c) Binding of mutated PfPTPA with PfPP2A in Xenopus oocytes. Co-immunoprecipitation experiment of the PfPTPA-PfPP2A complexes with anti-His antibodies (recognizing recombinant wild type and mutated PfPTPA) (upper panel) or with anti-Myc antibodies (recognizing PfPP2A) (lower panel) from microinjected Xenopus oocytes. Immunoprecipitates from Xenopus oocytes microinjected with water, WT or mutated PfPTPA were eluted and separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblot analysis was performed with anti-His or anti-Myc antibodies.
	Figure 4. Effect of wild type and mutated PfPTPA proteins on PP2A activity. The capacity of PfPTPA (WT or mutated) to regulate HuPP2A was assessed using pNPP activity tests. The human PP2A activity was measured at 405 nm by the release of p-nitrophenol after incubation with different concentration of recombinant WT or mutated PfPTPA proteins. Results presented as fold change in human PP2A activity are means ± SEM of three independent experiments (stars (*) represent significativity p = 0.01).
	Figure 5. Genetic studies of pfptpa and pfpp2a. (a,b) Gene-targeting construct for gene disruption by single homologous recombination using the pCAM-BSD, and the locus resulting from integration of the knockout pfptpa (a) or pfpp2a (b) construct. (c,d) Analysis of pCAM-BSD-pfptpa (c) and pCAM-BSD-pfpp2a (d) transfected 3D7 culture by PCR; lanes 1–3 correspond to DNA extracted from transfected parasites; lanes 4–6 correspond to DNA extracted from wild type parasites. Lanes 1 and 4 represent the detection of the full length wild type locus (PCR with p9-p8 and p31-p30 respectively); lanes 2 and 5 represent the detection of episomal DNA (PCR with p10 and p11); and lanes 3 and 6 represent the detection of integration of the insert (PCR with p9-p11 and p31-p11 respectively).
	Figure 6. Inhibition of G2/M transition of Xenopus oocytes by PfPTPA. (a) Percentage of GVBD induced by the progesterone (PG), PfPTPA or the microinjection of PfPTPA followed by PG incubation; (b) Percentage of GVBD induced by PG incubation after microinjection of different amount of PfPTPA; (c) Binding of PfPTPA with XePP2A in Xenopus oocytes. Co-immunoprecipitation experiment of the PfPTPA-XePP2A complex with anti-His antibodies (recognizing recombinant PfPTPA tagged with 6-His) (lanes 2 and 3) from microinjected Xenopus oocytes. The anti-mouse IgG antibody (lanes 1) was used as a control. Immunoprecipitates from Xenopus oocytes microinjected with water (−) or PfPTPA (+) were eluted, separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblot analysis was performed with anti-His antibodies (recognizing PfPTPA) (upper panel) or anti-XePP2A antibodies (lower panel); (d) Effect of PfPTPA on progesterone-dependent GVBD in Xenopus oocytes. Percentage of GVBD induced by PG after the microinjection of 10 ng of PfPTPA WT or mutated (n = 2).

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