Journal: bioRxiv

Article Title: Depolymerized lamins link nuclear envelope breakdown to mitotic transcriptional quiescence

doi: 10.1101/334110

Figure Lengend Snippet: a. pS22-LMNA and UnP-LMNA ChIP-seq signals at LADs. LADs are defined by UnP-LMNA ChIP-seq ( Methods ). A subset of LADs (885 of total 2,178) that do not have adjacent LADs within 250 kb downstream of LADs are shown. Inset shows location of strong ChIP-seq signals (marked in red). FE, fold enrichment. b. pS22-LMNA and UnP-LMNA ChIP-seq signals at 34,489 dLASs. c. Feature size of dLASs and LADs. d. Location of dLASs relative to LADs. e. Ty1 ChIP-seq signals at dLASs. Ty1 ChIP-seq was performed in BJ-5ta cells overexpressing Ty1-LMNA, Ty1-LMNC (Lamin C), phospho-deficient Ty1-LMNA-S22A/S392, or phospho-mimetic Ty1-LMNA-S22D/S392D. f. (Left) Fraction of dLASs overlapping anti-Ty1 ChIP-seq peaks in the overexpression cells shown in e . (Right) Fraction of anti-Ty1 ChIP-seq peaks overlapping dLASs. g. ATAC-seq, K27ac ChIP-seq, K4me3 ChIP-seq, and pS22-LMNA ChIP-seq signals at 73,933 ATAC-defined accessible sites in wild-type BJ-5ta cells.

Article Snippet: Antibodies used in ChIP are: rabbit monoclonal anti-phospho-Ser22-LMNA antibody D2B2E (Cell Signaling 13448S, Lot # 1; 5 µL per IP); mouse monoclonal anti-unphospho-Ser22-LMNA antibody E1 (Santa Cruz Biotechnology sc-376248, Lot # H2812; 10 µL per IP); mouse monoclonal anti-acetyl-Lys27 histone H3 antibody (Wako MABI0309, Lot # 14007; 2 µL per IP); mouse monoclonal anti-trimethyl-Lys4 histone H3 antibody (Wako MABI14004, Lot # 14004; 2 µL per IP); mouse monoclonal anti-Ty1 antibody (Diagenode C15200054, Lot # 005; 1 µL per IP).

Techniques: ChIP-sequencing, Over Expression

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

Journal: Nature Communications

Article Title: Allnighter pseudokinase-mediated feedback links proteostasis and sleep in Drosophila

doi: 10.1038/s41467-023-38485-7

Figure Lengend Snippet: a Micrograph of adult hemi-brain expressing UAS-mCD8-GFP under control of aln Ty1-T2A-Gal4 shows wide-spread expression of the aln gene in the brain and point to the layer of mechanosensory bristle neurons (arrowhead). Scale bar 40 µm. b , c Micrographs of adult heads expressing UAS-mCD8-GFP under control of aln Ty1-T2A-Gal4 and stained for Elav ( b’ ) or Dip-β ( c’ ) show expression of the aln gene in many neurons, including retinal mechanosensory bristle neurons and L4 lamina neurons (arrowheads in c ), but not photoreceptors. Enhanced example in white inset reveals dendrite and axon of mechanosensory bristle neuron. Scale bar in b is 40 µm, in ( c ) 20 µm. Experiments depicted in ( a – c ) were independently done 5 times. d , e Ty1-tagged Aln protein is detected by Western blot of lysates from adult heads of aln Ty1-T2A-Gal4 , LD, or LL treated as indicated and probed with antibodies against Ty1, w 1118 is used as negative control and Hook as loading control. e Quantification of blots for Ty1-tagged Aln relative to Hook protein ( n = 3). Data are from three experimental repeats. Statistical significance using two-tailed t test. Bar graphs show mean ± SD. f – h Endogenous Ty1-tagged Aln is detected in lamina and retina in micrographs of LD ( f , g ) or LL ( h ) treated adult heads with UAS-mCD8-GFP and without ( f ) or with aln Ty1-T2A-Gal4 driver ( g , h ) stained for Ty1 and GFP. Scale bar in ( f ) is 40 µm for ( f – h ). i – k Exogenous Flag-tagged Aln ( j ) or human Dipk1C ( k ) expressed under control of the aln Δ-Gal4 driver are detected in lamina and retina in micrographs of anti-Flag-stained adult heads, but not in wild-type control ( i ). Scale bar in ( i ) is 40 µm and is the same for ( i – k ). l – n In the retina, Ty1-tagged Aln partially colocalizes with Rab5-YFP ( l ), and Rab7 ( m ) but not Rab11-YFP ( n ). Scale bar in ( l ) is 5 µm for ( l – n ). Experiments depicted in ( f – h ) are repeated 3 times and l-n twice. Re: Retina, La: lamina, Me Medulla. Genotypes are listed in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: Antibody , Anti-Ty1 (mouse monoclonal, BB2) , Invitrogen , Thermo Fisher Scientific; MA5-23513 , (1:500 IHC, 1:2000 WB).

Techniques: Expressing, Staining, Western Blot, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: Allnighter pseudokinase-mediated feedback links proteostasis and sleep in Drosophila

doi: 10.1038/s41467-023-38485-7

Figure Lengend Snippet: a – c Ommatidial cross-sections show Ty1-tagged Aln in photoreceptor cell bodies ( a ) reduced levels in photoreceptors expressing QUAS-Shi ts1 ( b ) or w 1118 control ( c ) stained for chaoptin (24B10) and Ty1-Aln. Scale bar in ( a ) is 5 µm and is the same for ( a – c ). Numbers indicate n . d Quantification of Aln-Ty1 punctae per ommatidium. Bar graphs show mean ± SD, samples taken out of three experimental repeats. P -values for comparison to CarT>Shi ts1 ; aln Ty1-T2A-Gal4 from one-way ANOVA with Bonferroni correction for multiple comparisons are: w 1118 : 0.99; aln Ty1-T2A-Gal4 : <0.0001; F (2, 29) = 58.24. e , f Western blot of lysates from adult heads of w 1118 , aln Ty1-T2A-Gal4 ( n = 6) without or with CarT HA-T2A-QF2 -driven QUAS-Shi ts1 ( n = 4) expression. Quantification of blots for Ty1-tagged Aln relative to Hook protein ( f ). Data are from three experimental repeats. Two-tailed t test yielded P -value < 0.0001. Bar graphs show number of independent samples and mean ± SD. g – l Compared to controls ( g ), Ty1-Aln levels are reduced in response to CarT HA-T2A-QF2 -drived retinal expression of QUAS-Shi ts1 ( i , j ) or QUAS-TTL ( k , l ), especially in DIP-β marked L4 neurons (arrows in g’ – l’ ). Quantification of Aln-Ty1 punctae in L4 neurons identified by DIP-β staining ( m ) or in the retina ( n ), bar graphs show number of L4 neurons ( m ) or retinas ( n ) pooled from three independent experiments and mean ± SD. P -values for comparison to aln Ty1-T2A-Gal4 from one-way ANOVA with Tukey’s multiple comparisons test are ( m ) CarT>shi ts1 ; aln Ty1-T2A-Gal4 : <0.0001; CarT>TTL; aln Ty1-T2A-Gal4 : <0.0001; F = 131 ( n ) CarT>shi ts1 ; aln Ty1-T2A-Gal4 : <0.0001; CarT>TTL; aln Ty1-T2A-Gal4 : <0.0001; F = 86.1. o Compared to wild type, impaired photoreceptor synaptic activity in CarT HA-T2A-QF2 > QUAS-TTL, and CarT 43 null flies causes reduced photoreceptor structural plasticity as LL triggers loss of deep pseudopupil (DPP). Graphs show number of flies pooled from two independent experiments and means ± SD. p – s Aln expression is sensitive to disruption of the circadian rhythm as indicated by Aln-Ty1 levels in adult heads ( p – r ) exposed for 3 days to LD, LL or DD as indicated, ( s ) wild type controls. t Quantification of Aln-Ty1 punctae in retinas as shown in ( p – s ), bar graphs show median with interquartile range and number of independent sections pooled from two experiments. For comparison to LD aln Ty1-T2A-Gal4 , P -values from one-way ANOVA with Bonferroni correction for multiple comparisons are: LL: 0.0001; DD: 0.0001; w 1118 : 0.0012 ( F (3, 20) = 26.24). Scale bar in ( g ) is 100 µm and the same for ( g , i , k ). Scale bar in ( h ) is 50 µm and the same for ( h , j , l , p – s ). Significance threshold for P -Values show in ( d – f , m , n , t ): ns, non-significant; *<0.05; **<0.01; ***<0.001; ****<0.0001. Genotypes are listed in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: Antibody , Anti-Ty1 (mouse monoclonal, BB2) , Invitrogen , Thermo Fisher Scientific; MA5-23513 , (1:500 IHC, 1:2000 WB).

Techniques: Expressing, Staining, Western Blot, Two Tailed Test, Activity Assay

Journal: Nature Communications

Article Title: Allnighter pseudokinase-mediated feedback links proteostasis and sleep in Drosophila

doi: 10.1038/s41467-023-38485-7

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Ty1 (mouse monoclonal, BB2) , Invitrogen , Thermo Fisher Scientific; MA5-23513 , (1:500 IHC, 1:2000 WB).

Techniques: Generated

Journal: PLoS Pathogens

Article Title: Functional Analysis of the Leading Malaria Vaccine Candidate AMA-1 Reveals an Essential Role for the Cytoplasmic Domain in the Invasion Process

doi: 10.1371/journal.ppat.1000322

Figure Lengend Snippet: (A) Schematic representation of episomally expressed AMA-1 GFP fusion proteins. Signal peptide: blue; transmembrane domain: grey; pro-domain (P); domain I-III (DI-DIII) and cytoplasmic domain (CPD): brown; GFP: green. 3D7-derived AMA-1 is represented in lighter brown colour. Cleavage sites of the signal peptide and the pro-domain are marked with arrows. Sizes of the processed proteins are indicated either in green (GFP fusion) or in brown (endogenous protein). (B) Western blot analysis of transgenic parasites using anti-GFP antibodies recognize 3D7- and W2mef-derived AMA-1-GFP proteins (the 110 kDa and the processed 92 kDa AMA-1-GFP fusion protein) in the transgenic parasite lines but not in wild type (WT) parasites. (C) Western blot analysis of transgenic AMA-1 3D7 -GFP and AMA-1 W2mef -GFP expression using an AMA-1 specific polyclonal antibody. In wild type parasites, AMA-1 is recognized as an 83 kDa and a 66 kDa fragment (with and without pro peptide). In the transgenic cell lines, two additional proteins are apparent, representing the 110 kDa and the processed 92 kDa AMA-1-GFP fusion. (D) The monoclonal 1F9 antibody recognizes only the 3D7-derived but not the W2mef-derived AMA-1-GFP fusion protein. (E,F) Full-length AMA-1-GFP (green) localizes in schizonts (s) at the apical end of merozoites (m) and is distributed over the surface of merozoites after schizont rupture. This is shown in both unfixed (E) and fixed parasites (F). The distribution of the endogenous protein visualized with the 3D7-specific 1F9 antibody (red) is identical to the localization of the fusion protein (merge). (G,H) AMA-1-GFP dynamics during schizont rupture. (G) Time lapse microscopy of live AMA1-GFP–expressing parasites before and after schizont rupture. Strong apical GFP fluorescence with some surface staining was observed during schizont rupture and immediately after merozoite release (time frames from ). (H) This distribution is indistinguishable from AMA-1 in wild type parasites visualized by a polyclonal AMA1 antibody (green).

Article Snippet: Antibodies used in immunodetection were rabbit polyclonal anti AMA-1 anti AMA-1 monoclonal 1F9 , monoclonal anti TY1 (Diagenode) and monoclonal anti-GFP (Roche).

Techniques: Derivative Assay, Western Blot, Transgenic Assay, Expressing, Time-lapse Microscopy, Fluorescence, Staining

Journal: PLoS Pathogens

Article Title: Functional Analysis of the Leading Malaria Vaccine Candidate AMA-1 Reveals an Essential Role for the Cytoplasmic Domain in the Invasion Process

doi: 10.1371/journal.ppat.1000322

Figure Lengend Snippet: (A) Schematic representation of episomally expressed mutant AMA-1-GFP fusion proteins without cytoplasmic domain (AMA-1 Δtail -GFP). (B) Expression of mutant AMA-1-GFP was verified using anti-GFP specific antibodies. (C,D) The deletion of the cytoplasmic domain does not affect the localization of the AMA-1 Δtail -GFP fusion protein. This is shown in unfixed (C) and fixed (D) parasites. Mutant protein (green) colocalizes with the endogenous protein (red) as shown in the merge pictures. (E) Immunoprecipitation and immunoblot of AMA-1-GFP– interacting proteins. Coloured marker lines show the predicted size of the endogenous (brown), or the GFP-tagged AMA-1 (green). Supernatant (SN) of extract of AMA-1-GFP–expressing segmented schizonts was used on an AMA-1-GFP column. Supernatant (SN), flow through (FT), washing (W1-3), and the elution fraction (E) were separated on an SDS-PAGE gel and probed with polyclonal anti-PfAMA1 (PR) and anti-RON4.

Article Snippet: Antibodies used in immunodetection were rabbit polyclonal anti AMA-1 anti AMA-1 monoclonal 1F9 , monoclonal anti TY1 (Diagenode) and monoclonal anti-GFP (Roche).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Western Blot, Marker, SDS Page

Journal: Epigenetics & Chromatin

Article Title: The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

doi: 10.1186/s13072-018-0208-y

Figure Lengend Snippet: Histone acetylation prevents c-Myb binding to native K562 histones. a 18% SDS-PAGE of native histones isolated from K562 cells treated with DMSO (lane 1–3) or TSA (lane 4–6) and recombinant human histones loaded as reference (lane 7–9). b Acetylation of K562 histones was validated by western blotting using anti-H3K27ac primary antibody (ab177178, Abcam) and anti-H3 primary antibody (ab1791, Abcam) as reference. c GST pull-down assay performed with GST-fused c-Myb DBD (NR123) and 6 µg native K562 histone octamers. The western blot was analysed by using anti-H3 and anti-H3K27ac primary antibodies. About 10% (0.6 µg) of the K562 histones was loaded as reference

Article Snippet: The ChIP analysis was performed essentially as previously described [ , ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac.

Techniques: Binding Assay, SDS Page, Isolation, Recombinant, Western Blot, Pull Down Assay

Journal: Epigenetics & Chromatin

Article Title: The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

doi: 10.1186/s13072-018-0208-y

Figure Lengend Snippet: ChIP analysis of c-Myb occupancy and H3K27ac levels at the mim - 1 locus. a Map of the mim - 1 locus ( LECT2 leucocyte cell derived chemotaxin 2 ( Gallus gallus (Chicken) ). The enhancer mapped in is indicated. Myb recognition elements (MREs) are indicated by colour-coded asterisk (red: the strong MRE = YAACGG , green: MRE = YAACNG and blue: double MREs with palindromic orientation). The amplicons used for qRT-PCR quantification are indicated. b , c Occupancy of c-Myb wild-type (hcM) and D152V as measured by ChIP and qRT-PCR at the indicated regions. IgG IP controls are included. Bar graphs represent the mean ± SEM of three independent biological replicates each measured in triplicate. d Fragmentation of chromosomal DNA of one replicate. e – g Levels of H3K27ac as measured by ChIP and qRT-PCR at the indicated regions. Bar graphs represent the mean ± SEM of two independent biological replicates each measured in triplicate. ChIP was performed as described on HD11 cells 24 h after transfection

Article Snippet: The ChIP analysis was performed essentially as previously described [ , ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac.

Techniques: Derivative Assay, Quantitative RT-PCR, Transfection

Journal: Epigenetics & Chromatin

Article Title: The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

doi: 10.1186/s13072-018-0208-y

Figure Lengend Snippet: c-Myb-dependent transcriptional activation of a chromatin-embedded gene involves increased histone acetylation. HD-11 cells were transfected with plasmids encoding full-length HA-tagged c-Myb or c-Myb D152V. The cells were treated with 15 ng/ml TSA or DMSO 8 h after transfection. Total RNA was isolated 24 h after transfection, and mim - 1 and MYB expression was measured by qRT-PCR. The values of RNA expression were normalized to the relative amount of the reference gene hprt . The western blots were analysed using anti-c-Myb (ab45150, Abcam), anti-GAPDH (AM4300, Invitrogen) and anti-H3K27ac primary antibodies. The qRT-PCR results are presented as mean ± SEM of three independent biological replicates. Significance was evaluated by unpaired, two-tailed t tests on selected pairs and indicated with p values (* p < 0.05; ns p > 0.05). No p values were calculated for the MYB expression since only two biological replicates were used for this measurement

Article Snippet: The ChIP analysis was performed essentially as previously described [ , ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac.

Techniques: Activation Assay, Transfection, Isolation, Expressing, Quantitative RT-PCR, RNA Expression, Western Blot, Two Tailed Test

Journal: Epigenetics & Chromatin

Article Title: The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

doi: 10.1186/s13072-018-0208-y

Figure Lengend Snippet: Pioneer factor model. We propose a model for the pioneer factor function of c-Myb (left panel) where c-Myb binds to closed chromatin, possibly marked by H3K4me1, then recruits H3K27 acetyltransferases such as p300/CBP, facilitating histone acetylation. Due to the reduced nucleosomal stability caused by histone acetylation, chromatin accessibility increases and c-Myb detaches from chromatin because of the lost interaction with acetylated histones. The pioneer-defect D152V mutant (right panel) binds to chromatin and recruits H3K27 acetyltransferases, but is unable to facilitate H3K27ac due to the weakened histone interaction. Therefore, the D152V mutant is unable to increase chromatin accessibility, and the chromatin-embedded target gene remains silent

Article Snippet: The ChIP analysis was performed essentially as previously described [ , ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac.

Techniques: Mutagenesis