rabbit anti-ty1 antibodies  (GenScript corporation)

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: C-terminally tagged PfAlba2-Ty1, PfAlba3-Ty1, and PfAlba4-Ty1 were successfully expressed from an episome in P. falciparum asexual blood stages. ( A ) Schematic representation of the domain organization of the six Alba proteins of P. falciparum . ( B ) 3D7 parasites transfected with the pLN-PfAlba2-Ty1, pLN-PfAlba3-Ty1, or pLN-PfAlba4-Ty1 plasmids were grown in the presence of 5 mg/mL of blasticidin-S (BS), their genomic DNA harvested, and used with the indicated primers in PCRs to confirm the uptake of the episome. Primer pairs targeted either the genomic Alba locus ( p1 +p2 ) or different regions of the episome ( p3 +p4 and p5 +p6 ) as shown in the scheme ( top panel ), which is not drawn to scale. The DNA size marker corresponds to the GeneRuler 1 kb Plus DNA Ladder (Fermentas, Thermo Fisher Scientific). ( C ) PfAlba2-Ty1, PfAlba3-Ty1, or PfAlba4-Ty1 proteins were detected in protein lysates prepared from the indicated transfectant lines by Western blotting with mouse anti-Ty1 antibodies. PfHsp70 served as a loading control. ( D ) Immunofluorescence assays (IFAs) were used to determine the localization of PfAlba2-Ty1, PfAlba3-Ty1, and PfAlba4-Ty1 in ring (R), trophozoite (T), and schizont (S) stages of the indicated transfectant lines. Antibodies used included rabbit anti-Ty1 (red). Nuclei were labeled with DAPI (blue). Scale bar represents 5 µM. All of the cultures used for Western blotting and IFAs contained 5 µg/mL of blasticidin-S.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Transfection, Marker, Western Blot, Control, Immunofluorescence, Labeling

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: Overexpression of PfAlba2 or PfAlba3 adversely affects intra-erythrocytic growth of P. falciparum . ( A ) The growth of 3D7 + empty vector, 3D7 + PfAlba2-Ty1, 3D7 + PfAlba3-Ty1, and 3D7 + PfAlba4-Ty1 parasites was measured by flow cytometry for 5 days in the presence of the indicated concentrations of BS (in μg/mL). The y-axis denotes the percentage parasitemia at each time point. Data represent the means of a minimum of three independent experiments ± SEM (error bars). Red arrows indicate growth profiles of the strains at 10 µg/mL BS concentration. ( B ) The ~48-h IED cycle of the Alba-overexpressing lines was monitored by flow cytometry. The y-axis denotes the percentage of ring or schizont stages at each time point. Data represent the means of a minimum of three independent experiments ± SEM (error bars).

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Over Expression, Plasmid Preparation, Flow Cytometry, Concentration Assay

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: Transcriptomic data quality assessment by sample clustering demarcates the 3D7 + PfAlba2-ty1 and 3D7 + PfAlba3-Ty1 data sets from controls. ( A ) Schematic representation of the transcriptomics experiment. 3D7 + empty vector, 3D7 + PfAlba2-Ty1, 3D7 + PfAlba3-Ty1, or 3D7 + PfAlba4-Ty1 parasites were grown in white blood cell ( WBC )-free blood to ring (8–10 hpi) or trophozoite (28–30 hpi) stages in the presence of 5 µg/mL of blasticidin-S, total RNA harvested, and mRNA enriched and analyzed by strand-specific RNA-seq. Differential gene expression in the Alba-overexpressing lines relative to 3D7 and empty vector transfectants was quantified by DESeq2 analysis. ( B ) Principal component analysis of normalized read counts of 3D7, 3D7 + empty vector, 3D7 + PfAlba2-Ty1, 3D7 + PfAlba3-Ty1, and 3D7 + PfAlba4-Ty1 ring and trophozoite stage trancriptomes. Meaningful clustering of samples is indicated by dashed lines. ( C ) Pearson correlation coefficient R analysis of the normalized read counts from RNA-seq analysis of ring (R) and trophozoite (T) stage samples of the 3D7, 3D7 + empty vector, 3D7 + PfAlba2-Ty1, 3D7 + PfAlba3-Ty1, and 3D7 + PfAlba4-Ty1 strains. The color scale indicates the value of the R from 0 to 1. ( D ) Hours post-infection estimates for the transcriptomic data were obtained by passing the normalized RNA-seq data through the maximum likelihood algorithm developed by Lemieux et al . . The left panel shows the HPI estimates within 95% confidence intervals, while the right panel displays the actual likelihoods determined for each sample over the 48-h IED cycle.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Plasmid Preparation, RNA Sequencing, Gene Expression, Infection

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: PfAlba2 and PfAlba3 overexpression causes significant perturbations to the blood stage transcriptome. ( A ) Bar graph showing the total number of differentially expressed genes (DEGs) that are up- or downregulated in the indicated transgenic P. falciparum strain relative to controls. ( B ) Venn diagrams were used to represent the overlap in DEGs between the various Alba-overexpressing strains during ring and trophozoite stages. ( C ) Venn diagrams were used to represent the overlap in DEGs between ring and trophozoite stages of either 3D7 + PfAlba2-Ty1 or 3D7 + PfAlba3-Ty1 parasites. Troph = Trophozoite.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Over Expression, Transgenic Assay

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: Mistiming of key cellular developmental processes is linked to PfAlba3 overexpression. (A & B) Volcano plot of differentially expressed genes in ( A ) ring and ( B ) trophozoite stages of 3D7 + PfAlba3-Ty1 as compared to controls. Genes with |log 2 FC| > 1.5 and FDR < 0.05 values were considered to be differentially expressed (green dots). Pink dots indicate genes with |log 2 FC| > 1.5 but FDR > 0.05, purple dots indicate genes with |log 2 FC| < 1.5 but FDR < 0.05, and gray dots denote genes that are not changed significantly upon PfAlba3 overexpression. FC = fold change; FDR = false discovery rate. (C & D) Gene Ontology (GO) enrichment analysis in three categories, Cellular Component, Molecular Function, and Biological Process, for significantly up and downregulated genes in the ( C ) ring and ( D ) trophozoite stages of 3D7 + PfAlba3-Ty1 parasites. GO enrichment was performed after removing genes belonging to multigene families such as var and rifin . The number of genes enriched for each GO term relative to background is indicated on the right side of the y-axis , while the -log 10 (padj) of each GO term is represented on the left side of the y-axis . Note that p-adj is the same as FDR.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Over Expression

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: Global var transcriptional repression is apparent in the PfAlba2- and PfAlba3-overexpressing parasites. ( A ) var gene transcriptional profile in ring stages was assessed by RNA-seq of P. falciparum 3D7 transfected with either PfAlba2-Ty1, PfAlba3-Ty1, or PfAlba4-Ty1 expression plasmids. Empty vector (pLN-Ty1) transfectants served as a control. The steady-state mRNA levels of all 60 var genes is indicated in reads per kilobase of transcript per million or RPKM ( y-axis ). Two replicates for each sample were analyzed. ( B ) Total mRNA levels (in RPKM) of the var gene family was calculated for two ring-stage RNA-seq replicates of each indicated strain. Data represent the mean + STDEV.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: RNA Sequencing, Transfection, Expressing, Plasmid Preparation, Control

Journal: Genome Biology

Article Title: The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages

doi: 10.1186/s13059-015-0771-5

Figure Lengend Snippet: C-terminally tagged PfAlba1-Ty1 was successfully expressed from an episome in P. falciparum asexual blood stages. a The transfection and maintenance of the pPfAlba1-Ty1C plasmid was confirmed by PCR of genomic DNA. Primer pairs targeted endogenous ALBA1 ( p1+p2 ; see Additional file for primer sequences), the ALBA1-TY1 locus on the episome ( p3+p4 and p5+p6 ), and the unrelated ALBA4 genomic locus ( A4F+R ), in either untransfected 3D7 parasites or parasites harboring the empty vector or pPfAlba1-Ty1C. The scheme ( top panel ) is not drawn to scale; UTR = untranslated region. The leftmost lane ( bottom panel ) represents the size marker, GeneRuler 1 kb Plus DNA Ladder (Fermentas, Life Technologies). b PfAlba1-Ty1 was detected in protein lysates prepared from empty vector or PfAlba1-Ty1 parasites by denaturing gel electrophoresis and western blotting with mouse anti-Ty1 antibodies or anti-PfAlba1 antibodies. PfAldolase served as a loading control. c Immunofluorescence assays were used to determine the localization of PfAlba1-Ty1 in trophozoite ( T ) and schizont ( S ) stages of PfAlba1-Ty1 parasites. Empty vector transfectants served as a negative control. Antibodies used included mouse anti-Ty1 ( green ) or anti-PfAlba1 ( red ). Nuclei were labeled with DAPI ( blue ). Scale bar represents 1 μM

Article Snippet: The resulting parasite pellet was lysed under non-denaturing conditions in the presence of RNAsin (Promega) and subjected to immunoprecipitation analysis with rabbit anti-Ty1 antibodies or rabbit IgG antibodies (Sigma).

Techniques: Transfection, Plasmid Preparation, Marker, Nucleic Acid Electrophoresis, Western Blot, Immunofluorescence, Negative Control, Labeling

Journal: Genome Biology

Article Title: The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages

doi: 10.1186/s13059-015-0771-5

Figure Lengend Snippet: PfAlba1 overexpression reduces intra-erythrocytic growth of P. falciparum . a Protein lysates prepared from empty vector or PfAlba1-Ty1 transfectants grown in the presence of increasing concentrations of blasticidin-S ( BS ; 2.5–20 μg/ml) were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies, anti-PfAlba1 antibodies, or anti-PfAlba4 antibodies. PfAldolase served as a loading control. b The levels of PfAlba1-Ty1, as detected by the anti-Ty1 antibodies in ( a ), were measured by densitometry. For each BS concentration, after normalizing the PfAlba1-Ty1 signal to the PfAldolase signal, data were normalized to the 2.5 μg/ml sample. Data represent the means of a minimum of three independent experiments ± standard error (S.E.; error bars ). c The growth of empty vector ( left panel ) or PfAlba1-Ty1 ( right panel ) parasites was measured by flow cytometry for 5 days in the presence of the indicated concentrations of BS (in μg/ml). The y-axis denotes the percentage parasitemia at each time point. Data represent the means of a minimum of three independent experiments ± S.E. ( error bars ). d The ~48 h IDC of empty vector or PfAlba1-Ty1 transfectants was monitored by flow cytometry. The y-axis denotes the percentage of ring stages at each time point. The vertical dashed line represents one replication cycle. Data represent the means of three independent experiments ± S.E. ( error bars )

Article Snippet: The resulting parasite pellet was lysed under non-denaturing conditions in the presence of RNAsin (Promega) and subjected to immunoprecipitation analysis with rabbit anti-Ty1 antibodies or rabbit IgG antibodies (Sigma).

Techniques: Over Expression, Plasmid Preparation, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay, Flow Cytometry

Journal: Genome Biology

Article Title: The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages

doi: 10.1186/s13059-015-0771-5

Figure Lengend Snippet: In trophozoite stages, a PfAlba1-Ty1 ribonucleoprotein complex interacts with 1665 transcripts. a Schematic representation of the RNA immunoprecipitation protocol. Native lysates prepared from PfAlba1-Ty1 parasites were incubated with rabbit anti-Ty1 antibodies in the presence of protease and RNAse inhibitors to immunoprecipitate (IP) a PfAlba1-Ty1-containing ribonucleoprotein complex. The IPed RNA molecules were identified by strand-specific RNA-seq. RNA IPed from empty vector parasites served as background binding. b Lysates (10 % of the input used for immunoprecipitation) or proteins IPed with anti-Ty1 antibodies from empty vector or PfAlba1-Ty1 transfectants were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies or anti-PfAlba1 antibodies. PfAldolase served as a loading control. The contrasted image clearly shows the presence of PfAlba1 in the anti-PfAlba1-Ty1 co-IPed complex. c A Gaussian density kernel estimate of the distribution of gene ranks according to mRNA abundance of the 1665 transcripts that co-IPed with the PfAlba1-Ty1 complex. The y-axis represents relative density and the x-axis represents gene expression ranks, with 0 being least expressed. d Fourier phase distribution of the normalized RNA-seq data of the 1665 co-IPed transcripts. Top panel : P. falciparum IDC transcriptomic data were obtained from Bozdech et al . , where the authors provide a convenient metric, the Fourier phase, to define the hours post-infection ( HPI ) at which each gene is most abundantly transcribed. Bottom panel : a Gaussian density kernel estimate of the distribution of Fourier phase values of the 1665 transcripts that co-IPed with PfAlba1-Ty1 as sampled from Bozdech et al .

Article Snippet: The resulting parasite pellet was lysed under non-denaturing conditions in the presence of RNAsin (Promega) and subjected to immunoprecipitation analysis with rabbit anti-Ty1 antibodies or rabbit IgG antibodies (Sigma).

Techniques: Immunoprecipitation, Incubation, RNA Sequencing Assay, Plasmid Preparation, Binding Assay, Nucleic Acid Electrophoresis, Western Blot, Expressing, Infection

Journal: Genome Biology

Article Title: The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages

doi: 10.1186/s13059-015-0771-5

Figure Lengend Snippet: PfAlba1 overexpression perturbs the trophozoite stage transcriptome, resulting in the early onset of a schizont-like transcription profile. a Schematic representation of the transcriptomic profiling experiment. 3D7, 3D7+empty vector, or 3D7+PfAlba1-Ty1 parasites were grown in white blood cell ( WBC )-free blood to ring (8–10 h post-infection (h p.i.)) or trophozoite (28–30 h p.i.) stages, and RNA harvested and mRNA enriched and analyzed by strand-specific RNA-seq. Differential expression in 3D7+PfAlba1-Ty1 relative to 3D7 and empty vector transfectants was quantified by edgeR . b qRT-PCR analysis of the indicated genes was performed using total RNA isolated from empty vector or PfAlba1-Ty1 transfectants. The left , middle and right panels include genes that were upregulated, unchanged, or downregulated, respectively, according to RNA-seq. The y-axis denotes -ΔΔC t , calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error ( error bars ). c Hours post-infection ( HPI ) estimates for the transcriptomic data were obtained by passing the normalized RNA-seq data through the maximum likelihood algorithm developed by Lemieux et al . . The left panel shows the HPI estimates within 95 % confidence intervals, while the right panel displays the actual likelihoods determined for each sample over the 48 h IDC. d Giemsa-stained blood films showing ring ( R ), trophozoite ( T ) or schizont ( S ) stages of empty vector or PfAlba1-Ty1 transfectants. Insets show zoomed-in views of the indicated infected red blood cells

Article Snippet: The resulting parasite pellet was lysed under non-denaturing conditions in the presence of RNAsin (Promega) and subjected to immunoprecipitation analysis with rabbit anti-Ty1 antibodies or rabbit IgG antibodies (Sigma).

Techniques: Over Expression, Plasmid Preparation, Infection, RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Isolation, Staining

Journal: Genome Biology

Article Title: The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages

doi: 10.1186/s13059-015-0771-5

Figure Lengend Snippet: Eleven percent of the transcripts deregulated in PfAlba1-Ty1 trophozoites directly bind to PfAlba1, with binding resulting in the translational repression of select components of the erythrocyte invasion machinery. a Venn diagrams were used to represent the overlap of transcripts identified in the co-IPed ( co-IP ; Additional file ), in vitro -bound ( In Vitro ; Additional file ), and differentially regulated ( Exp. ; Additional file ) datasets. b Pie chart showing the functionally over-represented categories in the 105 PfAlba1-bound and deregulated transcripts. c Bootstrap hierarchical clustering was used to partition 14 transcripts upregulated in PfAlba1-Ty1 trophozoites based on their binding (log (fold change); blue shading) to PfAlba1-Ty1 ( co-IP ) and GST-PfAlba1 ( In Vitro ). The numbers in the boxes are the bootstrap scores. The resulting clusters were PfAlba1-bound ( upper cluster ) and PfAlba1-unbound (lower cluster). d Top panel : qRT-PCR analysis of the indicated genes was performed using mRNA isolated from empty vector or PfAlba1-Ty1 trophozoites. The y-axis denotes -ΔΔCt, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error ( error bars ). Bottom panel : protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies. Lysates prepared from either 3D7 rings ( R ctrl ) or schizonts ( S ctrl ) served as a control for protein detection, except for PfFIKK7.1. ** = Exceptions to the observed correlation between PfAlba1-RNA binding and translational repression

Article Snippet: The resulting parasite pellet was lysed under non-denaturing conditions in the presence of RNAsin (Promega) and subjected to immunoprecipitation analysis with rabbit anti-Ty1 antibodies or rabbit IgG antibodies (Sigma).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, In Vitro, Quantitative RT-PCR, Isolation, Plasmid Preparation, Nucleic Acid Electrophoresis, Western Blot, RNA Binding Assay

Journal: Genome Biology

Article Title: The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages

doi: 10.1186/s13059-015-0771-5

Figure Lengend Snippet: Translationally repressed transcripts associate with a PfAlba1 complex in trophozoites and are subsequently released in schizonts for efficient translation. a Schematic representation of the experimental setup. Native lysates prepared from PfAlba1-Ty1 parasites at the trophozoite (28–32 h) or schizont (42–45 h) stage were incubated with rabbit anti-Ty1 antibodies in the presence of protease and RNAse inhibitors to IP a PfAlba1-Ty1-containing ribonucleoprotein complex. IPed RNAs were extracted and analyzed by qRT-PCR. Finally, RNA association was compared with the presence of the corresponding protein in whole cell lysates. goi = gene of interest. b Lysates (20 % of the input used for immunoprecipitation) or proteins IPed with anti-Ty1 antibodies from PfAlba1-Ty1 trophozoite ( T ) or schizont ( S ) stages were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies or anti-PfAlba1 antibodies. PfAldolase served as a control. c Left panel : IPed RNA from PfAlba1-Ty1 trophozoite or schizont lysates was analyzed by qRT-PCR with primers specific to the indicated genes. The y-axis of the bar graph denotes percentage enrichment, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error ( error bars ). ** = The AMA1 transcript binds to PfAlba1 in trophozoite stages. Fold change of PfAlba1 association from trophozoites to schizonts, i.e. , T/S, was calculated as in “Materials and methods”. * = The association of PfAlba1 and Clag9 mRNA does not decrease from trophozoites to schizonts. Right panel : protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites ( T ) and schizonts ( S ) were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies

Article Snippet: The resulting parasite pellet was lysed under non-denaturing conditions in the presence of RNAsin (Promega) and subjected to immunoprecipitation analysis with rabbit anti-Ty1 antibodies or rabbit IgG antibodies (Sigma).

Techniques: Incubation, Quantitative RT-PCR, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot, Plasmid Preparation