Journal: EMBO Reports

Article Title: Dynamic expression of tRNA‐derived small RNAs define cellular states

doi: 10.15252/embr.201947789

Figure Lengend Snippet: A GO annotation of 109 common tsRNA‐interacting proteins. B Effects of candidate 5′‐tsRNAs: tsGlnCTG, tsGlyGCC, tsMetCAT, and tsIleGAT on uncapped luciferase mRNA in an in vitro translation system ( n = 2–3, error bars represent SEM, and significance was calculated using unpaired t ‐test). B′ Effects of tsGlnCTG on capped GFP mRNA in an in vitro translation system ( n = 2–3, error bars represent SD, and significance was calculated using unpaired t ‐test). C Polysome profiles of different stem vs. differentiating states tested in this study. Gray areas indicate the fractions collected for sequencing. C′ tsRNA distribution among the polysome fractions from indicated cell states. D Workflow to identify proteomic and transcriptomic interactions of tsGlnCTG in pluripotency vs. differentiating mESCs. E False discovery rate (FDR) of peptides detected in LC‐MS/MS of biotinylated tsGlnCTG pulldown. F Western blot showing differential interaction of Igf2bp1, Ybx1, and Rpl10 with tsGlnCTG between LIF and RA conditions. G Overlap between transcripts that are associated with tsRNAs in RA condition and the transcripts that are affected upon ASO transfection in RA conditions. H Schematic of the experimental setup to identify protein‐mediated and complementarity‐based tsRNA target interaction. I qPCR analysis showing the enrichment of transcripts pulled down by tsGlnCTG in lysates devoid of proteins compared with normal lysate ( n = 2, error bars represent SEM, and significance was calculated using unpaired t ‐test). J Top 10 tsGlnCTG‐associated proteins in indicated cell states. Data information: * P value < 0.05, ** P value < 0.01; *** P value < 0.001; n.s, non‐significant.

Article Snippet: 400 nM of tsGlnCTG was incubated with increasing concentrations (20, 40, 80, 200, and 400 nM) of recombinant Igf2bp1 (TP316226, Origene) for 30 min at 30°C.

Techniques: Luciferase, In Vitro, Sequencing, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transfection

Journal: EMBO Reports

Article Title: Dynamic expression of tRNA‐derived small RNAs define cellular states

doi: 10.15252/embr.201947789

Figure Lengend Snippet: A Merged triplicate Western immunoblots of Igf2bp1 and β‐actin in mESCs grown under LIF or RA conditions (top) and densitometric quantitation of the same (below) ( n = 3, error bars represent SD, and significance was calculated using Student's t ‐test). B Validation of siRNA‐mediated knockdowns of Igf2bp1 in RA‐treated mESCs ( n = 3, error bars represent SD, and significance was calculated using Student's t ‐test). B′ Expression of pluripotency genes in Igf2bp1 knockdown cells ( n = 3, error bars represent SD, and significance was calculated using Student's t ‐test). C Small RNAs identified (18–35 nt) from input and Igf2bp1 pulldown in RA‐treated mESCs. D qPCR validation of the tsGlnCTG enrichment upon Igf2bp1 pulldown in RA‐treated mESCs. E Binding curve showing the interaction of tsGlnCTG and Igf2bp1 (Kd = 33 nM). The intensities of the Igf2bp1 band were normalized to band intensity of the total Igf2bp1 protein used for pulldown. F Biological duplicate for c‐Myc quantitation from Igf2bp1 pulldown between LIF vs. RA conditions ( n = 2, for duplicate check Fig B). Data information: * P value < 0.05, ** P value < 0.01; *** P value < 0.001; n.s, non‐significant.

Article Snippet: 400 nM of tsGlnCTG was incubated with increasing concentrations (20, 40, 80, 200, and 400 nM) of recombinant Igf2bp1 (TP316226, Origene) for 30 min at 30°C.

Techniques: Western Blot, Quantitation Assay, Biomarker Discovery, Expressing, Knockdown, Binding Assay

Journal: EMBO Reports

Article Title: Dynamic expression of tRNA‐derived small RNAs define cellular states

doi: 10.15252/embr.201947789

Figure Lengend Snippet: In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. ( n = 2) error bars represent SEM. Quantification of the Igf2bp1‐bound c‐Myc mRNA between LIF‐ vs. RA‐treated mESCs ( n = 2; see Fig F for duplicate data). Association of c‐Myc transcript in different translating pools in RA‐treated mESCs as compared to LIF condition. ( n = 2), error bars represent SD, and significance was calculated by one‐tailed unpaired t ‐test. Relative enrichment of c‐Myc mRNA in translating (80S and polysome) and non‐translating (mRNPs) pools (fractionated from polysome profiling) between ASO‐treated and mock‐treated RA‐induced differentiating mESCs. ( n = 2), error bars represent SD, and significance was calculated using one‐tailed unpaired t ‐test. Relative levels of c‐Myc mRNA in ASO‐treated and mock‐treated RA‐induced differentiating mESCs compared with LIF‐treated mESCs. ( n = 3), error bars represent SD, and significance was calculated using one‐tailed unpaired t ‐test. Epistatic analysis of 5′‐tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity ( n = 3), error bars represent SD, and significance was calculated using unpaired t ‐test. Schematic representing tsRNA based c‐Myc transcript regulation. Data information: * P value < 0.05, ** P value < 0.01; *** P value < 0.001.

Article Snippet: 400 nM of tsGlnCTG was incubated with increasing concentrations (20, 40, 80, 200, and 400 nM) of recombinant Igf2bp1 (TP316226, Origene) for 30 min at 30°C.

Techniques: In Vitro, Binding Assay, One-tailed Test, Activity Assay

Journal: EMBO Reports

Article Title: Dynamic expression of tRNA‐derived small RNAs define cellular states

doi: 10.15252/embr.201947789

Figure Lengend Snippet: The dynamic expression of 5′‐tsRNAs plays a crucial role in modulating stem cell differentiation. During differentiation, 5′‐tsRNAs regulate the translation and/or the stability of several transcripts through its interaction with ribosomes, RNA‐binding proteins (ribonucleoproteins), such as IGF2BP1, or through direct sequence complementarity.

Article Snippet: 400 nM of tsGlnCTG was incubated with increasing concentrations (20, 40, 80, 200, and 400 nM) of recombinant Igf2bp1 (TP316226, Origene) for 30 min at 30°C.

Techniques: Expressing, Cell Differentiation, RNA Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

doi: 10.1093/nar/gkl575

Figure Lengend Snippet: Comparison of Zα domains ( A ) and schematic representation of constructs used for transfections ( B ) and protein expression ( C ). Comparison of Zα domains of human (hs) and mouse (mm) ZBP1, human ADAR1, zebrafish (dr) PKZ, vaccinia virus (vv) E3L and yaba-like disease virus (yldv) E3L is shown (A). The structures of the mouse (mm) Zα ZBP1 , human (hs) Zα ADAR1 , yaba-like disease virus (yldv) Zα E3L domains have been determined in complex with Z-DNA. Residues that make contact with Z-DNA, or the analogous residues in other Zα domains, are boxed in light blue. Asterisks mark the conserved asparagine and tyrosine residues that have been mutated in this study in hsZBP1, as well as a conserved tryptophan. Residues that form the hydrophobic core are boxed in green. Residues that are neither DNA contacting nor structural but match the consensus sequence are highlighted in yellow. Isoleucine 335 in Zβ ADAR1 is highlighted in red. (B) The exon composition of the most prominent ZBP1 splice variants ZBP1full and ZBP1ΔZα as well as that of artificial constructs are shown. Exon 7 is rarely found in mRNA. Exon 9 contains an alternative termination site . ZBP1full and ZBP1ΔZα have been expressed as un-tagged or GFP tagged proteins in HeLa cells. ZBP1ΔZβ, ZBP1ΔZαΔZβ, ZBP1E1-5 and ZBP1E1-5ΔZα were expressed as GFP-tagged proteins. Schematic representation of the exon composition of constructs expressed from pET28a (p28) vectors in E.coli are shown in (C).

Article Snippet: Recombinant Zαβ ZBP1 was immobilized on Aminolink matrix (Pierce) according to the manufacturer's instructions.

Techniques: Construct, Transfection, Expressing, Sequencing

Journal: Nucleic Acids Research

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

doi: 10.1093/nar/gkl575

Figure Lengend Snippet: SDS–PAGE analysis of recombinant proteins used for CD-spectrometry and EMSA. 500 nmol of each purified protein was subjected to SDS–PAGE and visualized by Coomassie Brilliant blue staining: Lane M, molecular mass marker; lane 1: Zαβ ADAR1 ; lane 2: Zα ZBP1 ; lane 3: Zβ ZBP1 ; lane 4: Zαβ ZBP1 ; lane 5: Zαβ ZBP1 N46D; lane 6: Zαβ ZBP1 Y50A; lane 7: Zαβ ZBP1 N46D/Y50A; lane 8: Zαβ ZBP1 N141D/Y145A; lane 9: Zαβ ZBP1 N46D/Y50A/N141D/Y145A.

Article Snippet: Recombinant Zαβ ZBP1 was immobilized on Aminolink matrix (Pierce) according to the manufacturer's instructions.

Techniques: SDS Page, Recombinant, Purification, Staining, Marker

Journal: Nucleic Acids Research

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

doi: 10.1093/nar/gkl575

Figure Lengend Snippet: Conformational change of poly(dC–dG) in the presence of Zα proteins in CD-spectrometry. The CD spectra show the titrations of Zα ZBP1 ( A ), Zβ ZBP1 ( B ), Zαβ ZBP1 ( C ) and Zαβ ADAR1 ( D ). The curves show DNA spectra in the absence (control, B-DNA) and in the presence of protein at protein/basepair molar ratio as labeled. Spectra were measured after 30 min of incubation at 24°C. ( E ) The CD spectra of wildtype (wt) Zαβ ZBP1 and quadruple mutant (Zαβ ZBP1 mut) carrying the four amino acid substitutions N46D, Y50A, N141D and Y145A are shown in relation to the control, where no protein was added. ( F ) Time dependent change of ellipticity at 255 nm wavelength over 30 min Zαβ ZBP1 and Zαβ ZBP1 mut are shown. Spectra are expressed in absolute values of ellipticity in millidegrees (mdeg).

Article Snippet: Recombinant Zαβ ZBP1 was immobilized on Aminolink matrix (Pierce) according to the manufacturer's instructions.

Techniques: Labeling, Incubation, Mutagenesis

Journal: Nucleic Acids Research

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

doi: 10.1093/nar/gkl575

Figure Lengend Snippet: Binding of Z-DNA by ZBP1 Zα proteins in electrophoretic mobility shift assays. EMSA were performed using 32 P-labeled double-stranded (dC–dG) 20 as probe in the presence of 20 000-fold access of sheared salmon sperm DNA as unspecific competitor. ( A ) Molar concentration was adjusted for the number of Zα domains the protein comprises. Zα and Zβ was assayed in twice as high molar concentration as Zαβ. Migration of the free probe is shown in lane 1 and 13. Zαβ (lane 2: 12.5 nM; lane 3: 25 nM; lane 4: 50 nM), Zα (lane 5: 25 nM; lane 6: 50 nM; lane 7: 100 nM), and Zβ (lane 5: 25 nM; lane 6: 50 nM; lane 7: 100 nM) were assayed at indicated concentrations. Reaction mixes shown in lanes 11–13 were prepared at the same time and were run on a separate TBE Gel. Zα and Zβ were mixed together at two concentrations (lane 11: 25 nM of each protein and lane 12: 50 nM of each protein) to see if there is a cooperative effect if both domains are present in different molecules. Sometimes a minor, slower migrating band was observed (asterisk) for the probe, probably due to a different secondary structure of the probe. ( B ) Zαβ ADAR1 (lane 2) and Zαβ ZBP1 (lane 3) form complexes with the radiolabeled probe whereas the indicated Zαβ ZBP1 mutants at equimolar concentration do not (lanes 4–8). Migration of the free probe is shown in lane 1.

Article Snippet: Recombinant Zαβ ZBP1 was immobilized on Aminolink matrix (Pierce) according to the manufacturer's instructions.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Concentration Assay, Migration

Journal: Nucleic Acids Research

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

doi: 10.1093/nar/gkl575

Figure Lengend Snippet: Different subcellular localization of two major ZBP1 splice variants. HeLa cells transiently transfected with ZBP1 were fixed and stained with polyclonal anti-ZBP1 antibodies and with secondary RPE-conjugated anti-rabbit-Ig antibodies. The left pictures show red fluorescence (RPE) after identical exposure time. The middle panels show nuclear stain using Hoechst33342. Right pictures show merges. In ( A ) no competitor was present, while in ( B ) 10 μg/ml of recombinant Zαβ ZBP1 was added. Anti-ZBP1 antibodies were used to stain for ZBP1 (left panels) in HeLa cells transfected with either full length ZBP1 ( C–E ) or the splice variant ZBP1ΔZα ( F–H ). Cells representative of three independent experiments are shown.

Article Snippet: Recombinant Zαβ ZBP1 was immobilized on Aminolink matrix (Pierce) according to the manufacturer's instructions.

Techniques: Transfection, Staining, Fluorescence, Recombinant, Variant Assay

Journal: Nucleic Acids Research

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

doi: 10.1093/nar/gkl575

Figure Lengend Snippet: Correlation of subcellular localization of ZBP1 with the capability of the first Zα domain to bind the Z-conformation. HeLa cells were transiently transfected with the indicated ZBP1-EGFP expression plasmids for 8–12 h. Subcellular localization was analyzed by fluorescence microscopy (left panels). Nuclei were stained with Hoechst33342 (middle panel). In ( A ) and ( B ) localization of the naturally occurring full length ZBP1 and ZBP1ΔZα, respectively, tagged with GFP is shown. Localization is shown for GFP-tagged full length ZBP1 with the indicated single ( C and D ), double ( E and F ) or quadruple ( G ) amino acid substitutions at residues important for Z-DNA binding. ( H and I ) show the localization of full length ZBP1 and ZBP1ΔZα proteins, respectively, after the precise deletion of exon 4, which encodes the entire second Zα domain. The localization of fusion proteins after deletion of the complete C-terminus (exons 6–10) is shown for ZBP1 ( J ) and ZBP1ΔZα ( K ). ( L ) shows the distribution of GFP alone within the cell. Two hours after transfection with full length ZBP1-GFP ( M ) and ZBP1ΔZα-GFP ( N ) constructs, cells were treated with Leptomycin B, an inhibitor of nuclear export, for 8 h prior to microscopy. Cells representative of at least three independent experiments are shown. ( O ) Western blot analysis shows that ZBP1-GFP fusion proteins were intact and not significantly degraded 10 h after transfection. Total cell lysates of transfected cells were subjected to SDS–PAGE and recombinant proteins were detected with an antibody directed against GFP. In lane 1 lysate of mock-transfected cells is shown as control. Lane 2 shows lysate of GFP transfected cells. The letters written in parenthesis for lanes 3–9 refer to the different fusion proteins that are shown in this figure as fluorescence pictures (A–K).

Article Snippet: Recombinant Zαβ ZBP1 was immobilized on Aminolink matrix (Pierce) according to the manufacturer's instructions.

Techniques: Transfection, Expressing, Fluorescence, Microscopy, Staining, Binding Assay, Construct, Western Blot, SDS Page, Recombinant

Journal: Nucleic Acids Research

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

doi: 10.1093/nar/gkl575

Figure Lengend Snippet: Association and dynamics of interaction of ZBP1 granules with SGs and PBs. HeLa cells were co-transfected with full length ZBP1-RFP ( A ), ZBP1ΔZα-RFP ( B and C ), full length ZBP1-GFP ( D ) and ZBP1ΔZα-GFP ( E ) plus SG markers G3BP-GFP (A and B), TIA1-GFP (C) or PB marker DCP1a-RFP (D and E). For each photo the indicated sections are shown in better resolution below for green, red and merged fluorescence. The dynamics of interaction between ZBP1 granules with SGs and PBs are shown for co-transfections with ZBP1ΔZα-RFP and TIA1-GFP ( F and Video 1), ZBP1ΔZα-RFP and G3BP-GFP (Video 2) or ZBP1ΔZα-GFP and DCP1a-RFP ( G and Videos 3 and 4). Cells were observed over a period of 15 min with pictures taken every 30 s. Pictures taken at 3 min intervals are shown in F and G. White arrows indicate ZBP1 granules that were unbound at the beginning of the observation period and later attached to SGs (F) or PBs (G). Yellow arrows indicate ZBP1 granules that stayed attached to SGs (F) or PBs (G) over the complete observation period. Cells representative of at least three independent experiments are shown.

Article Snippet: Recombinant Zαβ ZBP1 was immobilized on Aminolink matrix (Pierce) according to the manufacturer's instructions.

Techniques: Transfection, Marker, Fluorescence

Journal: Nucleic Acids Research

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

doi: 10.1093/nar/gkl575

Figure Lengend Snippet: Relocation of full length ZBP1 and ZBP1ΔZα upon arsenite treatment and heat shock. HeLa cells were co-transfected with G3BP-GFP and full ZBP1-RFP ( A – D ) or ZBP1ΔZα ( E – H ). After 7 h cells were incubated with 100 μg/ml emetine (B and F) or 500 μM arsenite for 1 h or exposed to heat shock at 44°C for 30 min (D and H). Upper panels show RFP fluorescence, middle panels GFP fluorescence and lower panels the merged views. Cells representative of at least three independent experiments are shown. Supplementary Data.

Article Snippet: Recombinant Zαβ ZBP1 was immobilized on Aminolink matrix (Pierce) according to the manufacturer's instructions.

Techniques: Transfection, Incubation, Fluorescence

Journal: Nucleic Acids Research

Article Title: Increased PTCHD4 expression via m 6 A modification of PTCHD4 mRNA promotes senescent cell survival

doi: 10.1093/nar/gkae322

Figure Lengend Snippet: IGF2BP1 binds to m6A-modified regions of PTCHD4 mRNA, promotes PTCHD4 mRNA stability in senescent cells. (A) RNA-binding proteins (RBPs) interacting with PTCHD4 mRNA in at least 4 different m6A-eCLIP-seq datasets (ENCORI database), sorted according to the number of identified binding sites; m6A readers are highlighted in blue. (B) WI-38 cells at ∼PDL40 were transfected for 6 h with siCtrl or siRNAs targeting individual m6A reader RBPs; 48 h later, total RNA was extracted and the levels of PTCHD4 mRNA were quantified by RT-qPCR analysis; the reduction in PTCHD4 mRNA levels after IGF2BP1 silencing is indicated (arrowhead). (C) WI-38 cells at ∼PDL22 or ∼PDL40 were transfected with siCtrl or siRNAs targeting IGF2BP1; 48 h later, total RNA was extracted and the levels of IGF2BP1 and PTCHD4 mRNAs were quantified by RT-qPCR analysis. (D) The stability of PTCHD4 mRNA was measured in WI-38 cells (∼PDL40) that had been transfected for 6 h with siCtrl or siIGF2BP1; 24 h later, cells were treated with 2 μg/ml Actinomycin D, total RNA was collected at the times shown, and mRNA levels were measured by RT-qPCR analysis and normalized to the level of 18S rRNA. Half-lives (t1/2) were estimated as the times needed for PTCHD4 mRNA to reach one-half (50%, discontinuous line) of its abundance at time 0 h in each group. MYC mRNA was included as a control labile transcript. (E) Binding of IGF2BP1 to PTCHD4 mRNA in WI-38 cells (∼PDL22 or ∼PDL40) was verified by RIP analysis. After RIP using IgG or anti-IGF2BP1 antibodies, the presence of PTCHD4 mRNA in the IP material was measured by RT-qPCR analysis and represented as percentage of the input. (F) Total RNA extracted from proliferating and senescent (IR-IS) WI-38 cells was subjected to m6A-eCLIP and RNA-seq analysis to identify m6A peaks on PTCHD4 mRNA. The Genome Browser tracks (using pyGenomeTracks) display the m6A-eCLIP-seq read distributions in PTCHD4 mRNA in proliferating and senescent WI-38 cells. The black dotted line indicates the region with the highest m6A enrichment in senescent cells compared to proliferating cells, located within the coding region (CDS1792-2366). Data are available at GEO (GSE247621). (G) m6A dot blot and RNA electrophoresis of biotinylated in vitro-transcribed RNA corresponding to the CDS1792-2366 of PTCHD4 mRNA, either unmodified or randomly methylated by addition of m6ATP in the reaction mix (A:m6A ratio of 4:1). The m6A dot blot, prepared with 200 ng RNA, was used to monitor the methylation of the in vitro-transcribed RNA. Equal loading of the transcribed RNA molecules was assessed by staining with methylene blue, and their integrity and purity were confirmed by visualization of the RNA after electrophoresis (‘RNA gel’). (H) Whole-cell lysates from WI-38 cells (∼PDL22 or ∼PDL40) were incubated with in vitro-transcribed, biotinylated RNAs corresponding to the CDS1792-2366 of PTCHD4 mRNA, either methylated or unmethylated; the complexes were pulled down using streptavidin beads, and the presence of IGF2BP1 among the bound proteins was determined by western blot analysis. Control proteins G3BP1 (predicted to not bind) and YTHDF2 (predicted to bind) were included. Input, 10 μg of total lysate used in the IP reaction. Data in (C–E) represent the means and S.D. from at least three biological replicates; statistical significance (*P< 0.05; **P< 0.01; ***P< 0.001) was established using Student's t-test. Other data are representative of three or more biological replicates.

Article Snippet: The fragmented RNA (2 μg) was then incubated with 100 ng of c-Myc-tagged IGF2BP1 human recombinant protein (Origene Technologies); after pulldown using anti-c-Myc-magnetic beads (Pierce, Thermo Fisher Scientific), IGF2BP1-bound RNA fragments were extracted and further assessed by RT-qPCR analysis.

Techniques: Modification, RNA Binding Assay, Binding Assay, Transfection, Quantitative RT-PCR, Control, RNA Sequencing Assay, Dot Blot, Electrophoresis, In Vitro, Methylation, Staining, Incubation, Western Blot

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal Anti-ZBP1 (H-9) , Santa Cruz Biotechnology , sc-271483; RRID: AB_10650130.

Techniques: Virus, Recombinant, Transfection, Staining, Western Blot, Electron Microscopy, Plasmid Preparation, Software, Microscopy

Journal: Cell host & microbe

Article Title: Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis

doi: 10.1016/j.chom.2021.05.009

Figure Lengend Snippet: (A) IB of L929, SVEC4–10, FLAG-ZBP1-reconstituted SVEC (WT ZBP1) and ZBP1-null SVEC (EV) (Sridharan et al., 2017) cells either left untreated (−) or pretreated with 100 U/mL of mouse IFN-β for 18 h (+) and either left uninfected (−) or infected at an MOI of 5 with E3Δ83N (+). L929 cells in rightmost panel were transfected with either a scrambled siRNA pool or an siRNA pool targeting ZBP1 for 36 h prior to IFN-β treatment. Lysates were harvested at 4 hpi, and, following SDS-PAGE, evaluated for phospho-MLKL and ZBP1.

Article Snippet: Cells were UV crosslinked at 5 hpi, lysed, and cell lysates were subjected to immunoprecipitation with either mouse anti-E3-specific antibody (BEI) or rabbit anti-FLAG antibody (cell signaling) specific for ZBP1 here).

Techniques: Infection, Transfection, SDS Page

Journal: Cell host & microbe

Article Title: Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis

doi: 10.1016/j.chom.2021.05.009

Figure Lengend Snippet: (A) Sequence alignment of the Za of VACV E3, variola E3, hADAR1 and mouse Zα1 and Zα2. Yellow signifies >70% sequence identity, amino acids with an asterisk above are known to be required for ZNA binding, and those in red have been targeted by point mutations (Kim et al., 2004, 2003). The GenBank accession numbers for the various sequences are as follows: GenBank: AAA02759 (vaccinia virus); GenBank: NP_042088 (variola virus); ADAR 1 (Homo sapiens): GenBank: AAB06697; ZBP1 (Mus musculus): GenBank: NP_067369.

Article Snippet: Cells were UV crosslinked at 5 hpi, lysed, and cell lysates were subjected to immunoprecipitation with either mouse anti-E3-specific antibody (BEI) or rabbit anti-FLAG antibody (cell signaling) specific for ZBP1 here).

Techniques: Sequencing, Binding Assay

Journal: Cell host & microbe

Article Title: Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis

doi: 10.1016/j.chom.2021.05.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were UV crosslinked at 5 hpi, lysed, and cell lysates were subjected to immunoprecipitation with either mouse anti-E3-specific antibody (BEI) or rabbit anti-FLAG antibody (cell signaling) specific for ZBP1 here).

Techniques: Produced, In Vitro, Generated, Recombinant, Staining, Transfection, Blocking Assay, Cell Viability Assay, Derivative Assay, Software, Flow Cytometry, Microscopy

Journal: eLife

Article Title: Elevated FBXO45 promotes liver tumorigenesis through enhancing IGF2BP1 ubiquitination and subsequent PLK1 upregulation

doi: 10.7554/eLife.70715

Figure Lengend Snippet:

Article Snippet: antibody , anti-IGF2BP1 (Rabbit Polyclonal) , Proteintech , Cat. #: 22803–1-AP;RRID: AB_2879173 , IHC (1:200).

Techniques: Knock-In, Isolation, Control, Recombinant, Immunoprecipitation, Mutagenesis, Software