EMBO/SourceData huggingface dataset:
Figure 7(A) HIV-1-infected primary CD4 T cells (strain CH058) were incubated with sera of HIV negative (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.(B) CH058-infected primary CD4 T cells were cultured for 24h with sera of healthy (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:100) and 50% NHS from a healthy donor. C3 surface levels on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.(C) CD4 T cells not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with the serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown on the left. On the right, results are expressed as MFI of staining. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Error bars indicate SEM. *P<0.05, Wilcoxon test. Only significant comparisons are depicted.(D) Primary CD4 T cells either not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with 50% NHS from a healthy donor and serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:100). The levels of C3 deposition on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown (left panel). Results are expressed as MFI of staining (right panel). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.(E) Correlation between patients' sera (sK104 and sKB105) antibody binding and C3 deposition on CD4 T cells either mock-infected or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058). Each dot is the mean of 6 donors of CD4 T cells. Correlation was analyzed by Spearman correlation. Correlation coefficient (r) and p-value are indicated.
Figure 1A T98G cells were challenged or not with dsDNAs or with 2'3'cGAMP for 6 h prior to whole cell extraction and Western Blot (WB) analysis using indicated antibodies.C Gli4 cells were treated as in A prior to WB analysis using indicated antibodies.F T98G cells were challenged or not with dsDNA for 6 h, in the presence or not of the NU7441 DNA-PKcs inhibitor, prior WB analysis using indicated antibodies.I T98G cells were treated or not with 0.16 µM camptothecin (CPT) for 48 h prior to immunofluorescence analysis using dsDNA- and 53BP1-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).J Whole cell extracts from T98G cells treated or not for 72 h with 0.16 µM CPT were analyzed by WB using indicated antibodies.
Figure 2A T98G cells were treated or not with dsDNA for 6 h prior to IF analysis using DNA-PKcs- and pDNA-PKcs-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).B Whole cell extracts from T98G cells were incubated with 80nt-long biotinylated ssDNA or dsDNA prior to pull-down using streptavidin-affinity beads. Input and eluates were analyzed by WB using indicated antibodies.C T98G cells were treated with non-targeting (CTRL) or KU70-targeting siRNAs prior to whole cell extract preparation, and pull-down as in B. Inputs and eluates were analyzed by WB using indicated antibodies.
Figure 3A T98G cells stably expressing cGAS (T98GcGAS) or not (T98GEmpty) were transfected or not with dsDNA for 6 h prior to whole cell extraction and WB analysis using indicated antibodies.C T98GEmpty and T98GcGAS were transfected or not with biotinylated dsDNA prior to whole cell extraction and pull-down using streptavidin-affinity beads. Inputs and eluates were analyzed by WB using indicated antibodies.J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.K THP-1 were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.
Figure 4F T98GEmpty and T98GcGAS stably expressing a GFP reporter (T98G-GFPEmpty and T98G-GFPcGAS, respectively) were xenotransplanted into the head of tg(mfap4:RFP) zebrafish line at 3 days post fertilization (dpf). Zebrafish embryos were imaged daily over 3 days. The graph represents the mean (±SEM) percentage of tumor growth normalized by the area on the day of transplantation (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).
Figure 5A Graph represents the mean (± SEM) fold migration of THP-1 cells through a 3 µm transwell insert when conditioned media from T98GEmpty or T98GcGAS was applied to lower chamber for 6 h. CCL2 was used as positive control (n=8 biological replicates).E Graph presents the quantification of macrophages recruited at tumor site 24 h post xenotransplantation in D (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).
Figure 6G Graph presents the survival rate of glioblastoma patients from A that present PRKDClow/MB21D1low, PRKDChigh/MB21D1low and PRKDChigh/MB21D1high expression.H Violin plots present the expression of PRKDC and MB21D1 in datasets analyzed in A, based on tumor grade (II to IV).
Figure 13D-brain model, horizontal brain section illustrating transgenic human P301Ltau expression in the entorhinal cortex (green, EC) of the ECrTgTaumouse lines, and the propagation of transgenic tau to the dentate gyrus (DG). Tau composition in ECrTgTau and control mouse lines investigated.Immunostained horizontal sections show the expression of human P301Ltau in ECneurons in absence of endogenous mousetau (ECrTgTau-Mapt0/0). Fluorescence in situ hybridization of humantau mRNA combined with immunofluorescent labeling (Immuno-FISH) of humantau protein (huTau) verifies P301Ltau transgene expression in the EC. Scale bars 50 μm.Propagation of humantau protein to neurons in the DG (white arrows) in ECrTgTau-Mapt0/0 mice. Close-ups show DG neurons from three ECrTgTau-Mapt0/0 mice (DG I-III). Immuno-FISH proofs the absence of humantau expression in DG neurons, which have huTau protein but no humantau mRNA. Scale bars 50 μm.Immunostained horizontal sections of ECrTgTaumice show the expression of human P301Ltau in ECneurons in presence of endogenous mousetau. Close-ups show DGneurons from three ECrTgTaumice (DG I-III) and Immuno-FISH proofs the absence of humantau expression in these DGneurons. Scale bars 50 μm.Human P301Ltau propagation to DG neurons (white arrows) in presence of endogenous mouse tau in ECrTgTau mice. Scale bars 50 µm.Human (huTau, antibody Tau13) and total tau (hu+moTau, DAKO) levels in entorhinal cortex (EC) extracts from 18-month-oldmice show equal human P301Ltau expression in ECrTgTau and ECrTgTau-Mapt0/0 mice (p=0.201, n=3 mice/group, one-way ANOVA with Bonferroni correction)The number of human tau-positive cell bodies in the DG (p=0.58, n=4 sections and 3 mice/group) and human tau in hippocampal (HPC) extracts (p=0.14, n=3 mice/group) were similar in ECrTgTau-Mapt0/0 and ECrTgTau mice (two-tailed Student's T-test).
Figure 2Primary cortical neuron cultures that were transduced with AAVeGFP-2a-P301Ltau at 7 DIV, and fixed and immunostained for GFP and humantau (Tau13 antibody) at 14 DIV, show tau donor (GFP+, huTau+; ~10% neurons) and a small number of tau recipient neurons (GFP-, huTau+; ~1% neurons). Western blot of whole cell lysates verified efficient cleavage (~95%) of eGFP and P301Ltau by the 2a peptide (n=3).Eight weeks after AAV injection into right EC of aged Mapt0/0 mice (=3), immunostained brain sections showed that huTauP301L (red) propagated to a few DG "recipient neurons" (white arrows). Scale bar 50 µm.Unilateral AAV-mediated human P301L tau expression in the EC and DG of age-matched WT mice (n=3). Representative images of brain sections show donor neurons in the injected EC and DG, and a few tau recipient neurons (white arrows) adjacent to the AAV injection site. Scale bar 50 µm.In the contralateral hemisphere of the same brain section, some tau recipient neurons (white arrows) were also present in the (non-injected) axonal projection areas in the contralateral EC (GFP-filled terminal ends). Scale bar 100 µm.
Figure 3A. Brain sections from 18-month-old ECrTgTau-Mapt0/0 and ECrTgTau mice were co-immuolabeled for human tau and misfolded tau (Alz50). Misfolded tau was only found in EC and DG neurons (white arrows) of ECrTgTau but not ECrTgTau-Mapt0/0 animals (n=4 sections/mouse, 3 mice/group). Scale bars 50 μm.B+C. Immunofluorescence labeling and stereological counting of microglia in entorhinal cortex (B) and astrocytes in hippocampus (C) indicated early signs of neurodegeneration in ECrTgTaumice. The significantly increased number of Iba1-positive microglia in the EC layer II/III of ECrTgtau mice (compared to WT) was partially rescued in ECrTgTau-Mapt0/0 mice (non-significant). The number of GFAP-positive astrocytes was similar across all genotypes (non-significant). Mean±SEM, n=4 sections per mouse, 3 mice/group, one-way ANOVA with Bonferroni correction. Scale bars 100 μm.
Figure 4Human, mouse and total tau protein levels in cortical TBS-extracts of rTg4510, rTg4510-Mapt0/0, and control mice: The amount of human tau (Tau13 antibody) was comparable in rTg4510 and rTg4510-Mapt0/0, moTau (Tau/5) was comparable in WT and rTg4510, and total tau levels (hu+moTau, DAKO antibody) were (expected) highest in rTg4510 mice. Mean±SEM, n=3 mice/group, non-significant.Whole brain weights of 9-month-old animals revealed pronounced brain matter loss in rTg4510 compared to WT mice (weight loss >16%), which was rescued in rTg4510-Mapt0/0 mice to >96%. Mean±SEM, n=5 mice/group.Cortical thickness measured adjacent to HPC, from CTX surface to corpus callosum, was decreased in rTg4510 mice by ≈25% compared to WT mice. rTg4510-Mapt0/0 showed no CTX thinning compared to Mapt0/0 or WT mcie. Mean±SEM, n=3 mice/group.The number of neurons (NeuN+ cells) in the cortex of rTg4510 mice was significantly reduced to ≈67% compared to both WT and rTg4510-Mapt0/0. Mean±SEM, n=3 mice/group.The volume of hippocampal region CA1, with CT the most effected regions in rTg4510 mice, was significantly reduced in rTg4510 by ~70% volume; rTg4510-Mapt0/0 had significantly larger CA1 volume left (reduced by only ~40%). Mean±SEM, n=3 mice/group.rTg4510 showed strong signs of neuroinflammation with extremely high numbers of activated astroglia (GFAP+, red) and microglia (Iba1+, white) in the CTX compared to WT mice. Both astro- and microgliosis was reduced by ~50% in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections/mouse and 5 mice per/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 µm.
Figure 5Cortical extracts from rTg4510-Mapt0/0 brains had significantly less phospho-tau (CP13, PHF1, 12E8) than rTg4510 extracts. Compared to WT mice, both transgenic tau lines had high levels of phospho-tau. Mean±SEM, n=3 mice/group.Representative images of gallyas silver stained aggregated tau in cortices from 12-month-old mice unravel stunning differences in the degree of tau pathology in rTg4510 compared to rTg4510-Mapt0/0 mice. Mean±SEM, n=3 mice/group.Higher magnification images of silver (12 month-old) and Thioflavine-S (9month-old) stained cortices show mature tangles (white arrows in Thio-S stain) in rTg4510 and rTg4510-Mapt0/0 mice; enhanced pathological changes such as neuritic tau accumulation and neuropil vacuolation around NFTs are found only in rTg4510 mice. Stereological counting revealed similar numbers of cortical NFTs between in rTg4510 and rTg4510-Mapt0/0 mice at 9 and 12 month of age. Because of the pronounced neuronal death only in rTg4510 mice, the percentage of tangle-bearing neurons was ≈1.6 to 1.8-fold higher higher in 9 and 12-month old rTg4510 mice. Mean±SEM, n=3 sections/mouse, 3 mice/group.Immuno-FISH for huTau mRNA (green) and phospho-tau (PHF1, red) shows obvious differences in the distribution of neurons in cortex layer II/III: 9-month-old rTg4510 mice had more neurons filled with NFT-like phosphorylated tau (PHF1+, red), and rTg4510-Mapt0/0 mice had significantly more huTau mRNA positive neurons (FISH+). rTg4510-Mapt0/0 mice had also more neurons still expressing both PHF1 and huTau mRNA (PHF1+ FISH+), suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections per mouse, 3 mice/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 μm.Immuno-FISH showing EC neurons having both misfolded somatic tau (Alz50, red) and human tau mRNA (green; white arrows) in rTg4510-Mapt0/0 but rarely in rTg4510 mice. n=3 sections, 2 mice/group. Scale bars 50 μm.
Figure 6Extraction of cortices revealed similar human tau (Tau13) in TBS-extracts (not significant) but significantly more human tau in TritonX-100 (TX-100) extracts of rTg4510-Mapt0/0 compared to rTg4510 mice. Mean±SEM, n =3 mice/group, two-tailed Student's T test, ns non significant.Native gel electrophoresis of cortical TBS-extracts showed small differences in HMW (oligomeric) human tau between rTg4510 and rTg4510-Mapt0/0 brains. Western blot lanes were averaged across ~2/3 of the width (black rectangular and arrow in Tau13 blot). The mean±SEM (n=3 mice/group) of these averages was plotted as longitudinal lane profiles. Differences in HMW tau are indicated by red and black arrows.TBS-brain extracts were applied to a HEK293 cell tau aggregation seeding assay(Holmes et al, 2014, Sanders et al, 2014), in which TauRDP301S-CFP and TauRDP301S-YFP are co-expressed intracellularly. The formation of intracellular fluorescent TauRDP301S aggregates leads to Foerster resonance energy transfer (FRET) activity between co-aggregated CFP and YFP-tags and correlates with the tau aggregation seeding activity of the applied brain extracts. After 24 hours, cells treated with extract (0.5 µg and 1.0 µg total protein per 96 well) from 9-month-old rTg4510 had significantly more intracellular YFP-positive (white arrows) aggregates compared to rTg4510-Mapt0/0 extracts; FRET activity of TauRDP301S aggregates appeared similar for both rTg4510 and rTg4510-Mapt0/0. WT and Mapt0/0 extracts never showed seeding activity. Addition of lipofectamine corrected for differences in cellular uptake of tau and led to similar differences in seeding activity between rTg4510 and rTg4510-Mapt0/0 mice. Two-tailed Student's T-test, mean±SEM ns, not significant, Insets 100 µm, Scale bars 50 µm.
Figure 2A-D LSM images of proximity ligation assay (PLA) dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. E-H LSM images of PLA dots composed of VEGFR2 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. I Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 9 embryos per genotype), *P = 0.022. J Quantification of the PLA dots indicating VEGFR2 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype). K Quantification of the total number of PLA dots indicating both VEGFR2 (left) and VEGFR3 (right) with phosphorylated tyrosine (p-Tyr) per jls/pTD section of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype), *P = 0.005 (VEGFR2/p-Tyr in ILK K.O. versus VEGFR3/p-Tyr in ILK K.O.), *P = 0.013 (VEGFR3/p-Tyr in control versus VEGFR3/p-Tyr in ILK K.O.)
Figure 3LSM images of cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 µmLSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1-positive area of control or ILK K.O. embryos (n = 6 embryos per genotype),
Figure 4Bright-field image of an E13.5 Flk1-Cre;Ilk∆/+;Itgb1∆/+ mouse embryo (referred to as "Control") with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jugular lymph sac / primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 µm, respectively. C, D Bright-field image of an E13.5 Flk1-Cre;Ilk∆/∆;Itgb1∆/+ embryo (referred to as "ILK & β1 integrin K.O.") with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jls/pTD. Scale bars: 500 and 100 µm, respectively. E-H LSM images of cross-sections through the jls/pTD of E13.5 control and ILK & β1 integrin K.O. embryos stained for the proliferation marker phospho-Histone H3. Arrows point to phospho-Histone H3-positive LECLSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on stained cross-sections through the jls/pTD of control and ILK & β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1 stained area. Scale bars: 10 µmNumber of LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryosLEC proliferation as determined by the number of phospho-Histone H3-positive LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryosQuantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK & β1 integrin K.O. embryos
Figure 5LSM images of a whole mount stained adult Prox1-CreERT2;Ilk+/+ mouse ear (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse ear (referred to as "Adult ILK K.O."), with higher magnification images indicated by dashed lines. Scale bars: 500 and LSM images of a whole-mount stained adult control and ILK K.O. ear, with higher magnification images indicated by dashed lines. Arrows point to proliferating LECs. Scale bars: 100 and 20 µm, respectivelyDermal lymph vessel density as determined by VEGFR3-positive area normalised to the total analysed area of adult control or ILK K.O. mice (n = 4 control and n = 5 ILK K.O. mice),LEC proliferation as determined by phospho-Histone H3-positive LECs normalised to analysed VEGFR3-positive area in the skin of adult control or ILK K.O. mice (with n = 7 control and n = 5 ILK K.O. mice),VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K.O. mice (n = 3 mice per genotype)LSM images of a whole mount stained adult control and ILK K.O. cornea, with detailed images on their right (L", M"). Arrows point to lymph vessels protruding into the cornea (encircled by a dotted line). Scale bars: 500 µm and 100 µm, re Number of lymph vessels in control or ILK K.O. corneas (n = 5 and n = 7 corneas),
Figure 6LSM images of cross-sections through the heart of an adult Prox1-CreERT2;Ilk+/+ mouse (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse (referred to as "Adult ILK K.O.") showing the outer lateral region of the left ventricle (LV) Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively (n = 4 control and n = 5 ILK K.O. mice)LSM images of cross-sections through the heart of an adult control and ILK K.O. mouse four weeks after myocardial ischemia and reperfusion (MI/R), showing the outer lateral region of the LV. Scale bars: 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively. (n = 5 control and n = 7 ILK K.O. mice), P = 0.051 (cardiac lymph vessel density in adult control versus adult ILK K.O.), *P = 0.025 (cardiac blood vessel density in adult control versus adult ILK K.O.)
Figure 7Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 µmLEC proliferation as determined by the number of BrdU-positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 3 independent transfections per siRNA), *P = 0.032 (control versus ILK-1), *P = 0.005 (control versus ILK-2), *P = 0.0003 (control versus ILK-3)VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 4 (control siRNA, ILK-1 siRNA, and ILK-3 siRNA), or n = 8 (ILK-2 siRNA) independent transfections per siRNA), *P = 0.0001 (control versus each siRNA)LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs (n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK-1), *P = 0.024 (control versus ILK-2), *P = 0.001 (control versus ILK-3)
Figure 8LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (-) mechanical stretch (n = 6 independent stretch chambers),Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 minutes, and used for immunoprecipitation (IP) of HA-tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (-) mechanical stretch; normalised to the respective amount of HA-tagged β1 integrin (n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers
Figure 1 Confocal images of HeLa cells stained with NF45/NF90 antibodies and antibodies against nucleolin and fibrillarin. Scale bar, 10 μm.  HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90.   Empty vector (EV), FLAG-NF45 plasmid (NF45) or FLAG-NF90 plasmid (NF90) were transfected into HEK293 cells for 24 h, and then rDNA-Luc was co-transfected into the cells. Luciferase activity was measured after a further 24 h (n=3). Western blots show the protein levels of FLAG-NF45 and FLAG-NF90.    E-F The qPCR analysis of 45S pre-rRNA from HEK293 cells treated with indicated shRNAs (E) and plasmids (F) for 24 h. Western blots show the expression levels of NF45, NF90, FLAG-NF45 and FLAG-NF90. (n = 3)
Figure 2  In Jurkat cells, a ChIP assay was performed with control IgG, NF45 and NF90 antibodies, and then the precipitated DNA was analyzed using qPCR with the aforementioned primers. The relative rDNA fold-enrichment was normalized to control IgG treatment. (n=3)  EMSA assay. Lane 1) Biotin-labled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section.  The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA -Luc wild type (WT) or mutated rDNA-Luc reporter plasmid (Mutant) for 24 h. (n=3) Western blot shows FLAG-NF90 protein level.