Journal: Scientific Reports

Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum

doi: 10.1038/s41598-017-12578-y

Figure Lengend Snippet: Strategies to investigate AP2-exp. ( a ) Predicted AP2-exp binding sites (vertical boxes) in 5′ UTRs of upsA, upsB and upsC var genes. ( b ) Top, AP2 domain (light gray box) of AP2-exp. Line shows the region used for obtention of specific antibodies. Bottom, western blot using anti-AP2-exp antibodies on cytoplasmic (C) and nuclear (N) extracts of early (ring) and late (troph) stages of 3D7 parasites. ( c ) Strategy used to disrupt the AP2-exp locus: the hdhfr cassette was inserted by double-crossover recombination. 1 and 2, 3 and 4 are oligonucleotides to detect recombination. Bellow, PCR on 3D7 wild-type (wt) and one mutant clone (mt 6). 5 kbp and 2 kbp molecular markers indicated on the left.

Article Snippet: Antibodies anti-AP2-exp were obtained from rats immunized with the recombinant GST-tagged N-terminal region of the AP2 protein using standard protocols by Genscript.

Techniques: Binding Assay, Western Blot, Mutagenesis

Journal: Scientific Reports

Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum

doi: 10.1038/s41598-017-12578-y

Figure Lengend Snippet: AP2-exp mutant parasites are viable and produce a short truncated protein. ( a ) One wild-type clone (circles, G7) and one mutant clone (triangles, MT6) were analysed by flow cytometry. ( b ) Single var genes expression profile on wild-type G7 clone and 2 AP2-exp mutants, MT6 and MT20. qRT-PCR was performed for individual var genes and relative copy number determined based on the seryl-tRNA synthetase gene. ( c ) Coverage of transcript reads (in reads per million, rpm) at the AP2-exp locus in the wild-type and mutant parasites at 30 hpi. Inset shows qRT-PCR confirming expression of homology region 1 in both MT6 and MT20. Below, translation of the disrupted locus in the mutants predicts a 33.6 kDa polypeptide. ( d ) Western blot of cytosolic and nuclear extracts from ring and trophozoite stages of G7 and MT6 parasites using anti-AP2-exp. Cytosol and nuclear controls were anti-Pfaldolase and anti-core human H3, respectively. Cropped bands are displayed and full-length membrane is in Supplementary Figure .

Article Snippet: Antibodies anti-AP2-exp were obtained from rats immunized with the recombinant GST-tagged N-terminal region of the AP2 protein using standard protocols by Genscript.

Techniques: Mutagenesis, Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot, Membrane

Journal: Scientific Reports

Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum

doi: 10.1038/s41598-017-12578-y

Figure Lengend Snippet: Differential gene expression in the mutant parasites. ( A ) Genes belonging to multigene families up- or down-regulated in the AP2-exp mutant parasites. Family names are indicated followed by the total number of differentially-expressed genes. ( B ) Box-and-whiskers plot of RNA-seq analysis of mutant parasites. Log2 fold-change in mutants versus wild-type is shown.

Article Snippet: Antibodies anti-AP2-exp were obtained from rats immunized with the recombinant GST-tagged N-terminal region of the AP2 protein using standard protocols by Genscript.

Techniques: Gene Expression, Mutagenesis, RNA Sequencing

Journal: Scientific Reports

Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum

doi: 10.1038/s41598-017-12578-y

Figure Lengend Snippet: AP2-exp mutant causes upregulation of surface protein expression and morphological changes in the host cell. ( A ) Western blot using antibodies against the conserved C-terminus of RIFINs on extracts of G7, MT6, MT20, and a positive control, FCR3S1.2 (+). Loading control, anti-hsp70. ( B ) Western blot using anti-STEVOR on uninfected RBC (RBC), G7 and MT6. Loading controls: anti-glycophorin ( c ) and histone H3. Cropped bands are shown and full-length membranes are available in Supplementary Figure . ( c ) Scanning electron microscopy of RBCs infected with G7 and MT6. Scale bar, 1 μm.

Article Snippet: Antibodies anti-AP2-exp were obtained from rats immunized with the recombinant GST-tagged N-terminal region of the AP2 protein using standard protocols by Genscript.

Techniques: Mutagenesis, Expressing, Western Blot, Positive Control, Control, Electron Microscopy, Infection