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Santa Cruz Biotechnology c myc dtx3l
C Myc Dtx3l, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/c+myc+dtx3l/DTX3L+Antibody/pm26479788-521-57-59
Average 94 stars, based on 11 article reviews

c myc dtx3l - by Bioz Stars, 2026-09

94/100 stars

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Transfection:

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.
Article Snippet: For co-immunoprecipitation of histone H2BJ with PARP9-DTX3L-STAT1, HEK293T cells were transfected for 48 h to express hemagglutinin (HA)tagged H2BJ (Life Technologies), FLAG-tagged PARP9 and c-Myc-tagged DTX3L, and cell lysates were immunoprecipitated with anti-HA (HA-7; Sigma) or c-Myc (E6654; Sigma) and were analyzed by immunoblot with antibody to HA-H2BJ (H6908; Sigma), antibody to FLAG-PARP (M2; Sigma), antibody to c-Myc–DTX3L (9E10; Santa Cruz) and antibody to STAT1 (1; BD Biosciences). .. For co-immunoprecipitation of histone H2BJ with PARP9-DTX3L-STAT1, HEK293T cells were transfected for 48 h to express hemagglutinin (HA)tagged H2BJ (Life Technologies), FLAG-tagged PARP9 and c-Myc-tagged DTX3L, and cell lysates were immunoprecipitated with anti-HA (HA-7; Sigma) or c-Myc (E6654; Sigma) and were analyzed by immunoblot with antibody to HA-H2BJ (H6908; Sigma), antibody to FLAG-PARP (M2; Sigma), antibody to c-Myc–DTX3L (9E10; Santa Cruz) and antibody to STAT1 (1; BD Biosciences). .. Cell lysates were incubated with anti-c-Myc (E6654, Sigma), and immunoprecipitated DNA was analyzed by real-time PCR assay with primers for the human IFIT1 promoter sequence (5′-CAGCAGGAATTCCGCTAGC-3′ and 5′-GCCAATGGTGTAAGCTGTGG-3′ as the forward and reverse primer pair, and 5′-GTGTGTCCTTGCAAGTTGG-3′ as the probe).

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection
Article Snippet: For X-ChIP protein interaction assays, cell lysates were sheared by a 29-gauge syringe (instead of by sonication) and were incubated with agarose beads conjugated to antibody to p300 (sc-584; Sigma), then were washed and analyzed by immunoblot with anti-STAT1, anti-PARP9, anti-DTX3L or anti-p300 (all identified above). .. For co-immunoprecipitation of histone H2BJ with PARP9-DTX3L-STAT1, HEK293T cells were transfected for 48 h to express hemagglutinin (HA)-tagged H2BJ (Life Technologies), FLAG-tagged PARP9 and c-Myc-tagged DTX3L, and cell lysates were immunoprecipitated with anti-HA (HA-7; Sigma) or c-Myc (E6654; Sigma) and were analyzed by immunoblot with antibody to HA-H2BJ (H6908; Sigma), antibody to FLAG-PARP (M2; Sigma), antibody to c-Myc–DTX3L (9E10; Santa Cruz) and antibody to STAT1 (1; BD Biosciences). ..

Immunoprecipitation:

Western Blot:

Image Search Results

( a ) Expression (log 2 normalized) of mRNA from pancreatic tissue of CAG-STAT1 and CAG-STAT1-CC mice at baseline, from a whole-genome array. Each symbol represents an individual gene: magenta, white and blue indicate genes expressed differentially in CAG-STAT1 mice versus CAG-STAT1-CC mice (antiviral function, key); gray indicates genes not expressed differentially; labels indicate genes ( n = 25) with the greatest increase in expression. ( b ) Expression of mRNA from U3A-STAT1 and U3A-STAT1-CC cells at baseline (presented as in a ); red indicates genes expressed differentially in both transgenic tissue and U3A cell lines (overlap, bottom right). Top left, retroviral vectors for U3A cell lines. ( c ) Expression (as in a , b ) of mRNA from U3A-STAT1 and U3A-STAT1-CC cells treated for 1 d with IFN-β (10 U/ml), presented relative to expression at baseline. TG, transgenic tissue. ( d ) Expression of RNA encoding EMCV 3D (assessed 1 d after infection with EMCV (MOI, 1)), the IAV polymerase P3 (assessed 2 d after infection with IAV (MOI, 1)) or the SINV RNA-dependent RNA polymerase Nsp4 (assessed 1 d after infection with SINV (MOI, 10)) in U3A, U3A-STAT1, and U3A-STAT1-CC cells stably transduced with lentivirus encoding control shRNA or shRNA specific for PARP9 or DTX3L (two shRNAs for each (shRNA-1 and shRNA-2); horizontal axes). * P < 0.05, versus U3A cells, and ** P < 0.05, versus U3A-STAT1-CC cells transduced with control shRNA (unpaired t -test). Data are from one experiment with pooling of three mice or samples ( a – c ) or are representative of three independent experiments ( d ; mean and s.e.m.).

Journal: Nature Immunology

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

doi: 10.1038/ni.3279

Figure Lengend Snippet: ( a ) Expression (log 2 normalized) of mRNA from pancreatic tissue of CAG-STAT1 and CAG-STAT1-CC mice at baseline, from a whole-genome array. Each symbol represents an individual gene: magenta, white and blue indicate genes expressed differentially in CAG-STAT1 mice versus CAG-STAT1-CC mice (antiviral function, key); gray indicates genes not expressed differentially; labels indicate genes ( n = 25) with the greatest increase in expression. ( b ) Expression of mRNA from U3A-STAT1 and U3A-STAT1-CC cells at baseline (presented as in a ); red indicates genes expressed differentially in both transgenic tissue and U3A cell lines (overlap, bottom right). Top left, retroviral vectors for U3A cell lines. ( c ) Expression (as in a , b ) of mRNA from U3A-STAT1 and U3A-STAT1-CC cells treated for 1 d with IFN-β (10 U/ml), presented relative to expression at baseline. TG, transgenic tissue. ( d ) Expression of RNA encoding EMCV 3D (assessed 1 d after infection with EMCV (MOI, 1)), the IAV polymerase P3 (assessed 2 d after infection with IAV (MOI, 1)) or the SINV RNA-dependent RNA polymerase Nsp4 (assessed 1 d after infection with SINV (MOI, 10)) in U3A, U3A-STAT1, and U3A-STAT1-CC cells stably transduced with lentivirus encoding control shRNA or shRNA specific for PARP9 or DTX3L (two shRNAs for each (shRNA-1 and shRNA-2); horizontal axes). * P < 0.05, versus U3A cells, and ** P < 0.05, versus U3A-STAT1-CC cells transduced with control shRNA (unpaired t -test). Data are from one experiment with pooling of three mice or samples ( a – c ) or are representative of three independent experiments ( d ; mean and s.e.m.).

Article Snippet: For expression of PARP9 and DTX3L, cDNA encoding FLAG-tagged human PARP9 or PARP9-M12 or c-Myc- tagged DTX3L or DTX3LM (Invitrogen) was inserted into lentiviral vector pLvx-IRES-puro (Clonetech) or pRRLsin.hCMV.IRES.EGFP to generate pLvx-FLAG-PARP9-IRES-puro, pLvx-c-Myc-DTX3L-IRES-Puro and pRRL-PARP9-IRES-GFP.

Techniques: Expressing, Transgenic Assay, Infection, Stable Transfection, Transduction, shRNA

Viral RNA (as in ) in U3A-STAT1, U3A-STAT1-PARP9, U3A-STAT1-DTX3L and U3A-STAT1-PARP9-DTX3L cells (key) with (− IFN-β) or without (+ IFN-β) pretreatment for 6 h with various doses IFN-β (horizontal axes), assessed 1 d after infection with EMCV, 2 d after infection with IAV (strain A/WS/33) or 1 d after infection with SINV. * P < 0.05, versus U3A-STAT1 cells (unpaired t -test). Data are representative of three independent experiments (mean and s.e.m.).

Journal: Nature Immunology

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

doi: 10.1038/ni.3279

Figure Lengend Snippet: Viral RNA (as in ) in U3A-STAT1, U3A-STAT1-PARP9, U3A-STAT1-DTX3L and U3A-STAT1-PARP9-DTX3L cells (key) with (− IFN-β) or without (+ IFN-β) pretreatment for 6 h with various doses IFN-β (horizontal axes), assessed 1 d after infection with EMCV, 2 d after infection with IAV (strain A/WS/33) or 1 d after infection with SINV. * P < 0.05, versus U3A-STAT1 cells (unpaired t -test). Data are representative of three independent experiments (mean and s.e.m.).

Techniques: Infection

$( a ) Immunoblot analysis of the co-immunoprecipitation of FLAG-tagged STAT1 (left and middle) or STAT1-CC (right) with PARP9 or DTX3L (left margin) in parental U3A cells (left) or U3A cells expressing FLAG-tagged STAT1 (middle) or STAT1-CC (right), with (γ or β) or without (−) treatment for 24 h with IFN-γ (100 U/ml) or IFN-β (1,000 U/ml) (above lanes). ( b ) Immunoblot analysis of tyrosine-phosphorylated STAT1 (pY-STAT1), serine-phosphorylated STAT1 (pS-STAT1), or total STAT1, PARP9 or DTX3L (left margin) in cytosolic (C) and nuclear (N) fractions of U3A, U3A-STAT1 or U3A-STAT1-CC cells with or without treatment for 0.3 h with IFN-γ (as in a ). Dashed outlines indicate tyrosine-phosphorylated STAT1, PARP9 and DTX3L in the nuclear fraction. ( c ) Immunostaining of U3A-STAT1-PARP9-DTX3L cells with (+ LMB) or without treatment for 1 h with leptomycin B (10 ng/ml), then with (+ IFN-β) or without (− IFN-β) treatment for 0.3 h with IFN-β (1,000 U/ml) plus leptomycin B. Scale bar, 10 μm. ( d ) Crosslinking ChIP assay of the binding of STAT1 to the IRF1 promoter in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells left untreated (− IFN-β) or treated (+ IFN-β) for 0.3 h with IFN-β; after incubation of lysates with antibody to STAT1, the immunoprecipitated DNA was analyzed by real-time PCR with primers for IRF1 promoter, then results were normalized to those of input DNA. Results obtained with control antibody (mouse immunoglobulin G (IgG)) were similar to those of cells transfected with empty vector only (data not shown). ( e ) Transactivation of ISRE and GAS in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells transfected for 12 h with a luciferase reporter vector for ISRE (left) or GAS (right) and left untreated (−) or treated (+) with IFN-β (left) or IFN-γ (right); results are presented as luminescence units (LU) relative to the activity of renilla luciferase. ( f ) Translocation of STAT1 to the nucleus in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells before (−) and after (+) treatment for 0.3 h with IFN-β (1,000 U/ml) (left) or IFN-γ (100 U/ml) (right), presented as fluorescence units (FU) (nuclear fluorescence − cytoplasmic fluorescence); initial negative values (for −IFN-β) indicate that STAT1 was mainly cytoplasmic. * P < 0.01, versus U3A-STAT1 cells (unpaired t -test). Data are representative of three independent experiments (mean and s.e.m. ( d , e ) or mean and s.e.m. of three wells with 500 cells per well ( f )).$

Journal: Nature Immunology

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

doi: 10.1038/ni.3279

Figure Lengend Snippet: ( a ) Immunoblot analysis of the co-immunoprecipitation of FLAG-tagged STAT1 (left and middle) or STAT1-CC (right) with PARP9 or DTX3L (left margin) in parental U3A cells (left) or U3A cells expressing FLAG-tagged STAT1 (middle) or STAT1-CC (right), with (γ or β) or without (−) treatment for 24 h with IFN-γ (100 U/ml) or IFN-β (1,000 U/ml) (above lanes). ( b ) Immunoblot analysis of tyrosine-phosphorylated STAT1 (pY-STAT1), serine-phosphorylated STAT1 (pS-STAT1), or total STAT1, PARP9 or DTX3L (left margin) in cytosolic (C) and nuclear (N) fractions of U3A, U3A-STAT1 or U3A-STAT1-CC cells with or without treatment for 0.3 h with IFN-γ (as in a ). Dashed outlines indicate tyrosine-phosphorylated STAT1, PARP9 and DTX3L in the nuclear fraction. ( c ) Immunostaining of U3A-STAT1-PARP9-DTX3L cells with (+ LMB) or without treatment for 1 h with leptomycin B (10 ng/ml), then with (+ IFN-β) or without (− IFN-β) treatment for 0.3 h with IFN-β (1,000 U/ml) plus leptomycin B. Scale bar, 10 μm. ( d ) Crosslinking ChIP assay of the binding of STAT1 to the IRF1 promoter in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells left untreated (− IFN-β) or treated (+ IFN-β) for 0.3 h with IFN-β; after incubation of lysates with antibody to STAT1, the immunoprecipitated DNA was analyzed by real-time PCR with primers for IRF1 promoter, then results were normalized to those of input DNA. Results obtained with control antibody (mouse immunoglobulin G (IgG)) were similar to those of cells transfected with empty vector only (data not shown). ( e ) Transactivation of ISRE and GAS in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells transfected for 12 h with a luciferase reporter vector for ISRE (left) or GAS (right) and left untreated (−) or treated (+) with IFN-β (left) or IFN-γ (right); results are presented as luminescence units (LU) relative to the activity of renilla luciferase. ( f ) Translocation of STAT1 to the nucleus in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells before (−) and after (+) treatment for 0.3 h with IFN-β (1,000 U/ml) (left) or IFN-γ (100 U/ml) (right), presented as fluorescence units (FU) (nuclear fluorescence − cytoplasmic fluorescence); initial negative values (for −IFN-β) indicate that STAT1 was mainly cytoplasmic. * P < 0.01, versus U3A-STAT1 cells (unpaired t -test). Data are representative of three independent experiments (mean and s.e.m. ( d , e ) or mean and s.e.m. of three wells with 500 cells per well ( f )).

Techniques: Western Blot, Immunoprecipitation, Expressing, Immunostaining, Binding Assay, Incubation, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Translocation Assay, Fluorescence

( a ) Domains of PARP and DTX3L (top; amino acid positions above and right), and structure (below) of the two macro domains (Macro1 and Macro2) in PARP9, for binding of ADP-ribose (left), and the RING domain in DTX3L, for E3 ligase activity (right), based on homology models, identifying glycine residues (left) or cysteine zinc-chelating residues (right) targeted for loss-of-function substitution. Models based on the crystal structure of histone macroH2A1.1 (Protein Data Bank accession code, 1ZR3 ) with bound ADP-ribose (left) or the structure of the equine herpesvirus-1 RING domain (Protein Data Bank accession code, 1CHC ) (right). ( b ) Gene expression (log 2 normalized) in U3A-STAT1-PARP9-DTX3L cells versus U3A-STAT1-PARP9M-DTX3LM cells, from whole-genome array, presented relative to expression in U3A-STAT1 cells; red indicates genes expressed differentially here whose expression was increased by treatment with IFN-β (as reported in ). ( c ) ISG expression in U3A-STAT1, U3A-STAT1-PARP9-DTX3L, U3A-STAT1-PARP9M-DTX3LM, U3A-STAT1-PARP9-DTX3LM and U3A-STAT1-PARP9M-DTX3L cells (key) left untreated (− IFN-β) or treated (+ IFN-β) for 12 h with IFN-β (10 U/ml). ( d ) CHART PCR assays of cells as in c . ( e ) Viral RNA (as in ) in U3A-STAT1 cell lines given no pretreatment (− IFN-β) or pretreated with various concentrations (horizontal axes) of IFN-β (+ IFN-β) and then inoculated with EMCV (left), IAV (middle) or SINV (right). ( f ) Significance of histone ubiquitination ( P value) versus ubiquitination of various histones (key) with DTX3L relative to their ubiquitination without DTX3L (horizontal axis), assessed by ubiquitin proteome array with DTX3L as the E3 ligase; dashed lines indicate a P value of 0.05 and a change in ubiquitination of '0-fold', so results in the top right quadrant are both statistically significant ( P < 0.05) and biologically relevant (change in ubiquitination of >0-fold). ( g ) Native ChIP assay of the binding of ubiquitinated histone H2B (H2B-Ub), histone H3 trimethylated at Lys4 (H3K4me3) or H2B to the IFIT1 promoter in various U3A cell lines (as in c ) left untreated (− IFN-β) or treated (+ IFN-β) for 16 h with IFN-β (1 U/ml), followed by incubation of nucleosomes from the cells with antibody to ubiquitinated H2B, trimethylated H3K4 or H2B, then real-time PCR analysis of the IFIT1 promoter sequence in the immunoprecipitated DNA; results were normalized to those of the corresponding input DNA and are presented relative to those of U3A-STAT1 cells, set as 1. Results obtained with control antibody (mouse IgG) were similar to those of cells transfected with empty vector only (data not shown). * P < 0.05, versus U3A-STAT1 cells (unpaired t -test). Data from one experiment with pooling of three cell samples ( b , f ) or are representative of three independent experiments ( c – e , g ; mean and s.e.m.).

Journal: Nature Immunology

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

doi: 10.1038/ni.3279

Figure Lengend Snippet: ( a ) Domains of PARP and DTX3L (top; amino acid positions above and right), and structure (below) of the two macro domains (Macro1 and Macro2) in PARP9, for binding of ADP-ribose (left), and the RING domain in DTX3L, for E3 ligase activity (right), based on homology models, identifying glycine residues (left) or cysteine zinc-chelating residues (right) targeted for loss-of-function substitution. Models based on the crystal structure of histone macroH2A1.1 (Protein Data Bank accession code, 1ZR3 ) with bound ADP-ribose (left) or the structure of the equine herpesvirus-1 RING domain (Protein Data Bank accession code, 1CHC ) (right). ( b ) Gene expression (log 2 normalized) in U3A-STAT1-PARP9-DTX3L cells versus U3A-STAT1-PARP9M-DTX3LM cells, from whole-genome array, presented relative to expression in U3A-STAT1 cells; red indicates genes expressed differentially here whose expression was increased by treatment with IFN-β (as reported in ). ( c ) ISG expression in U3A-STAT1, U3A-STAT1-PARP9-DTX3L, U3A-STAT1-PARP9M-DTX3LM, U3A-STAT1-PARP9-DTX3LM and U3A-STAT1-PARP9M-DTX3L cells (key) left untreated (− IFN-β) or treated (+ IFN-β) for 12 h with IFN-β (10 U/ml). ( d ) CHART PCR assays of cells as in c . ( e ) Viral RNA (as in ) in U3A-STAT1 cell lines given no pretreatment (− IFN-β) or pretreated with various concentrations (horizontal axes) of IFN-β (+ IFN-β) and then inoculated with EMCV (left), IAV (middle) or SINV (right). ( f ) Significance of histone ubiquitination ( P value) versus ubiquitination of various histones (key) with DTX3L relative to their ubiquitination without DTX3L (horizontal axis), assessed by ubiquitin proteome array with DTX3L as the E3 ligase; dashed lines indicate a P value of 0.05 and a change in ubiquitination of '0-fold', so results in the top right quadrant are both statistically significant ( P < 0.05) and biologically relevant (change in ubiquitination of >0-fold). ( g ) Native ChIP assay of the binding of ubiquitinated histone H2B (H2B-Ub), histone H3 trimethylated at Lys4 (H3K4me3) or H2B to the IFIT1 promoter in various U3A cell lines (as in c ) left untreated (− IFN-β) or treated (+ IFN-β) for 16 h with IFN-β (1 U/ml), followed by incubation of nucleosomes from the cells with antibody to ubiquitinated H2B, trimethylated H3K4 or H2B, then real-time PCR analysis of the IFIT1 promoter sequence in the immunoprecipitated DNA; results were normalized to those of the corresponding input DNA and are presented relative to those of U3A-STAT1 cells, set as 1. Results obtained with control antibody (mouse IgG) were similar to those of cells transfected with empty vector only (data not shown). * P < 0.05, versus U3A-STAT1 cells (unpaired t -test). Data from one experiment with pooling of three cell samples ( b , f ) or are representative of three independent experiments ( c – e , g ; mean and s.e.m.).

Techniques: Binding Assay, Activity Assay, Expressing, Incubation, Real-time Polymerase Chain Reaction, Sequencing, Immunoprecipitation, Transfection, Plasmid Preparation

( a ) PARP9, DTX3L and STAT1 constructs (left half of each side) used for the identification of interacting domains, with tagging of epitopes with FLAG, c-Myc or hemagglutinin (HA) (key) and inclusion or deletion of various domains (construct designations, left margins); fold and secondary structure prediction were used for the identification of reasonable boundaries for domains D1, D2 and D3 of DTX3L. R, RING domain; CC, coiled-coil domain; SH2, Scr-homology 2 domain; TA, transactivation domain; del, deletion. Right half (of each side), domain interactions. ( b – e ) Immunoblot analysis (IB) of protein complexes immunoprecipitated (IP) together in lysates of HEK293T cells co-expressing various combinations of the constructs in a (above lanes), identifying the following binding (dashed outlines): STAT1-HA with c-Myc–DTX3L domains ( b ), STAT1-HA with FLAG-PARP9 domains ( c ), c-Myc–DTX3L with FLAG-PARP9 domains ( d ), and FLAG-PARP9 with c-Myc–DTX3L domains ( e ); arrowheads immunoprecipitated protein. Below, control immunoblot analysis with the same antibody used for immunoprecipitation and immunoblot. ( f ) Constructs for PARP9-M12 and DTX3L-delD3 (top), and immunoprecipitation and immunoblot analysis (below) of U3A-STAT1 cells transfected with vector alone (far left lanes) or vector expressing various constructs (above lanes), as well as a control immunoblot for protein input (far right); β-actin serves as a loading control throughout. ( g ) Construct for DTX3L-RDTX (top), and immunoblot analysis (below) of U3A-STAT1 cell lines transduced with vector alone (far left lane) or vector expressing various constructs (above lanes), assessed 6 h after infection with EMCV. ( h ) Expression of mRNA from the ISGs IFIT1 , IFIT3 and OASL in U3A-STAT1 cell lines transduced with various constructs (key), then left untreated or treated for 12 h with IFN-β (10 U/ml). Data are representative of three independent experiments (mean and s.e.m. in h ).

Journal: Nature Immunology

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

doi: 10.1038/ni.3279

Figure Lengend Snippet: ( a ) PARP9, DTX3L and STAT1 constructs (left half of each side) used for the identification of interacting domains, with tagging of epitopes with FLAG, c-Myc or hemagglutinin (HA) (key) and inclusion or deletion of various domains (construct designations, left margins); fold and secondary structure prediction were used for the identification of reasonable boundaries for domains D1, D2 and D3 of DTX3L. R, RING domain; CC, coiled-coil domain; SH2, Scr-homology 2 domain; TA, transactivation domain; del, deletion. Right half (of each side), domain interactions. ( b – e ) Immunoblot analysis (IB) of protein complexes immunoprecipitated (IP) together in lysates of HEK293T cells co-expressing various combinations of the constructs in a (above lanes), identifying the following binding (dashed outlines): STAT1-HA with c-Myc–DTX3L domains ( b ), STAT1-HA with FLAG-PARP9 domains ( c ), c-Myc–DTX3L with FLAG-PARP9 domains ( d ), and FLAG-PARP9 with c-Myc–DTX3L domains ( e ); arrowheads immunoprecipitated protein. Below, control immunoblot analysis with the same antibody used for immunoprecipitation and immunoblot. ( f ) Constructs for PARP9-M12 and DTX3L-delD3 (top), and immunoprecipitation and immunoblot analysis (below) of U3A-STAT1 cells transfected with vector alone (far left lanes) or vector expressing various constructs (above lanes), as well as a control immunoblot for protein input (far right); β-actin serves as a loading control throughout. ( g ) Construct for DTX3L-RDTX (top), and immunoblot analysis (below) of U3A-STAT1 cell lines transduced with vector alone (far left lane) or vector expressing various constructs (above lanes), assessed 6 h after infection with EMCV. ( h ) Expression of mRNA from the ISGs IFIT1 , IFIT3 and OASL in U3A-STAT1 cell lines transduced with various constructs (key), then left untreated or treated for 12 h with IFN-β (10 U/ml). Data are representative of three independent experiments (mean and s.e.m. in h ).

Techniques: Construct, Western Blot, Immunoprecipitation, Expressing, Binding Assay, Transfection, Plasmid Preparation, Transduction, Infection

( a ) Immunoblot analysis of EMCV 3C in various U3A (left) or U3A-STAT1 (right) cell lines (above lanes) at 6 h after infection with EMCV (MOI, 1). ( b ) Immunoblot analysis of EMCV 3C and PSMB8 in various U3A cell lines (above lanes) left untreated (−) or treated (+) for 1 or 2 h (below blot) with the PSMB8-specific inhibitor ONX-0914 (0.1 μM) and then infected for 6 h with EMCV (MOI, 1). ( c ) EMCV RNA in cells as in a . ( d ) Immunoblot analysis of various proteins (left margin) in various U3A-STAT1 cell lines (above lanes) treated for 6 h with IFN-β (10 U/ml) or not (below blot) and then infected for 18 h with EMCV (MOI, 1). ( e ) Immunoblot analysis of EMCV 3C and other proteins (left margin) in HEK293T cells transfected for 36 h with various vectors (above lanes) alone (−) or together with (+) vectors encoding histidine- and V5-tagged EMCV 3C (His-V5-EMCV-3C) plus histidine-tagged ubiquitin (His-Ub) and then treated for 14 h with MG-132 (20 μM) (+MG-132) or not (−MG-132) (below blots). ( f ) Immunoblot analysis of EMCV 3C in human tracheal epithelial cells infected with EMCV at an MOI of 1 or 10 (top) and then, at 32 h later, treated for 12 h with MG-132 (20 μM) (+) or not (−). ( g ) EMCV 3D pol RNA in cells as in f . ( h ) Immunoblot analysis of primary cultures of human tracheal epithelial cells with or without treatment for 24 h with interferon (above lanes), showing the co-immunoprecipitation of endogenous STAT1 with PARP9-DTX3L (top), and input controls (below). Data are representative of three independent experiments (mean and s.e.m. in c , g ).

Journal: Nature Immunology

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

doi: 10.1038/ni.3279

Figure Lengend Snippet: ( a ) Immunoblot analysis of EMCV 3C in various U3A (left) or U3A-STAT1 (right) cell lines (above lanes) at 6 h after infection with EMCV (MOI, 1). ( b ) Immunoblot analysis of EMCV 3C and PSMB8 in various U3A cell lines (above lanes) left untreated (−) or treated (+) for 1 or 2 h (below blot) with the PSMB8-specific inhibitor ONX-0914 (0.1 μM) and then infected for 6 h with EMCV (MOI, 1). ( c ) EMCV RNA in cells as in a . ( d ) Immunoblot analysis of various proteins (left margin) in various U3A-STAT1 cell lines (above lanes) treated for 6 h with IFN-β (10 U/ml) or not (below blot) and then infected for 18 h with EMCV (MOI, 1). ( e ) Immunoblot analysis of EMCV 3C and other proteins (left margin) in HEK293T cells transfected for 36 h with various vectors (above lanes) alone (−) or together with (+) vectors encoding histidine- and V5-tagged EMCV 3C (His-V5-EMCV-3C) plus histidine-tagged ubiquitin (His-Ub) and then treated for 14 h with MG-132 (20 μM) (+MG-132) or not (−MG-132) (below blots). ( f ) Immunoblot analysis of EMCV 3C in human tracheal epithelial cells infected with EMCV at an MOI of 1 or 10 (top) and then, at 32 h later, treated for 12 h with MG-132 (20 μM) (+) or not (−). ( g ) EMCV 3D pol RNA in cells as in f . ( h ) Immunoblot analysis of primary cultures of human tracheal epithelial cells with or without treatment for 24 h with interferon (above lanes), showing the co-immunoprecipitation of endogenous STAT1 with PARP9-DTX3L (top), and input controls (below). Data are representative of three independent experiments (mean and s.e.m. in c , g ).

Techniques: Western Blot, Infection, Transfection, Immunoprecipitation

( a ) Immunostaining of EMCV 3C, DTX3L, and STAT1 in U3A-STAT1-PARP9M-DTX3LM cells at 5 h after infection with EMCV (MOI, 1). Scale bar, 10 μm. ( b ) Immunoblot analysis of the co-immunoprecipitation of EMCV 3C with PARP9-DTX3L in lysates of various U3A and U3A-STAT1 cell lines (above lanes) incubated for 5 min at 25 °C with recombinant histidine- and V5-tagged EMCV 3C (His-V5-EMCV-3C), followed by immunoprecipitation with antibody to (anti-) V5, probed with anti-c-Myc or anti-FLAG. ( c ) Crosslinking ChIP assay of various U3A-STAT1 cell lines without infection (−ECMV) or 6 h after infection with EMCV (MOI, 1) (+ ECMV) (key); after incubation of lysates with antibody to c-Myc–DTX3L, the immunoprecipitated DNA was analyzed by real-time PCR assay for IFIT1 promoter sequence, and results were normalized to input DNA and those of U3A-STAT1 cells. Results obtained with control antibody (mouse IgG) were similar to those of U3A-STAT1 cells (data not shown). ( d ) Immunoblot analysis of the ubiquitination of EMCV 3C in reaction mixtures containing various combinations (above lanes) of histidine- and V5-tagged EMCV (0.5 μg) as substrate, plus hemagglutinin-tagged ubiquitin (HA-Ub), E1 (ubiquitin-activating enzyme), the E2 ubiquitin-conjugating enzyme HbcH5a and DTX3L, detected with antibody to V5-tagged EMCV 3C and anti-hemagglutinin. The smear of bands with higher molecular weight indicates ubiquitinated proteins; native (non-ubiquitinated) histidine- and V5-tagged EMCV 3C monomer migrates as a 22-kDa protein. ( e ) Immunoblot analysis of the ubiquitination of EMCV 3C as in c , but with the addition of various amounts (above lanes) of histidine-tagged PARP9 and DTX3L to the reaction mixtures. ( f ) Immunoblot analysis of the ubiquitination of recombinant histidine- and V5-tagged tagged EMCV 3C after incubation with E1, E2 and DTX3L, with (+) or without (−) hemagglutinin-tagged ubiquitin, analyzed directly (−Ni-NTA) or after purification by nickel–nitrilotriacetic acid chromatography (+Ni-NTA), probed with anti-hemagglutinin and anti-V5. ( g ) Immunoblot analysis of the ubiquitination of HRV 3C in an assay as in f . ( h ) Immunoblot analysis of the K48-linked ubiquitination of EMCV 3C in an assay as in f , but with incubation with K48-linked ubiquitin (K48-Ub), and probed with antibody to K48-linked ubiquitin. ( i ) Immunoblot analysis of the ubiquitination of EMCV 3C and respiratory syncytial virus NS1 (RSV NS1) in the assay conditions in f . ( j ) Ubiquitination of EMCV 3C by DTX3L, DTX3L-delD3 and DTX3L-RDTXH in the assay conditions in f . Arrowheads ( f – h,j ) indicate ubiquitinated forms. ( k ) Binding of DTX3L-RING-DTXH to EMCV 3C (top) or HRV 3C (bottom), with DTX3L-RING-DTXH concentrations of 10, 5, 2.5, 1.25 and 0.625 μM (for EMCV 3C) or 8.5, 4.25, 2.13, 1.06 and 0.53 μM (for HRV 3C), assessed by real-time biolayer interferometry and presented as sensorgrams (left); solid lines indicate reference-corrected data. Right, orientation of biosensor pin. ( l ) Ubiquitination of EMCV 3C in various HEK293T cells transfected with various constructs (above lanes), assessed by immunoprecipitation of lysates with anti-V5 beads and then with anti-V5 in solution, followed by immunoblot analysis with antibody to hemagglutinin-tagged ubiquitin and anti-V5. * P < 0.05, versus uninfected cells (unpaired t -test). Data are representative of at least three independent experiments (mean and s.e.m. in c ).

Journal: Nature Immunology

Article Title: PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

doi: 10.1038/ni.3279

Figure Lengend Snippet: ( a ) Immunostaining of EMCV 3C, DTX3L, and STAT1 in U3A-STAT1-PARP9M-DTX3LM cells at 5 h after infection with EMCV (MOI, 1). Scale bar, 10 μm. ( b ) Immunoblot analysis of the co-immunoprecipitation of EMCV 3C with PARP9-DTX3L in lysates of various U3A and U3A-STAT1 cell lines (above lanes) incubated for 5 min at 25 °C with recombinant histidine- and V5-tagged EMCV 3C (His-V5-EMCV-3C), followed by immunoprecipitation with antibody to (anti-) V5, probed with anti-c-Myc or anti-FLAG. ( c ) Crosslinking ChIP assay of various U3A-STAT1 cell lines without infection (−ECMV) or 6 h after infection with EMCV (MOI, 1) (+ ECMV) (key); after incubation of lysates with antibody to c-Myc–DTX3L, the immunoprecipitated DNA was analyzed by real-time PCR assay for IFIT1 promoter sequence, and results were normalized to input DNA and those of U3A-STAT1 cells. Results obtained with control antibody (mouse IgG) were similar to those of U3A-STAT1 cells (data not shown). ( d ) Immunoblot analysis of the ubiquitination of EMCV 3C in reaction mixtures containing various combinations (above lanes) of histidine- and V5-tagged EMCV (0.5 μg) as substrate, plus hemagglutinin-tagged ubiquitin (HA-Ub), E1 (ubiquitin-activating enzyme), the E2 ubiquitin-conjugating enzyme HbcH5a and DTX3L, detected with antibody to V5-tagged EMCV 3C and anti-hemagglutinin. The smear of bands with higher molecular weight indicates ubiquitinated proteins; native (non-ubiquitinated) histidine- and V5-tagged EMCV 3C monomer migrates as a 22-kDa protein. ( e ) Immunoblot analysis of the ubiquitination of EMCV 3C as in c , but with the addition of various amounts (above lanes) of histidine-tagged PARP9 and DTX3L to the reaction mixtures. ( f ) Immunoblot analysis of the ubiquitination of recombinant histidine- and V5-tagged tagged EMCV 3C after incubation with E1, E2 and DTX3L, with (+) or without (−) hemagglutinin-tagged ubiquitin, analyzed directly (−Ni-NTA) or after purification by nickel–nitrilotriacetic acid chromatography (+Ni-NTA), probed with anti-hemagglutinin and anti-V5. ( g ) Immunoblot analysis of the ubiquitination of HRV 3C in an assay as in f . ( h ) Immunoblot analysis of the K48-linked ubiquitination of EMCV 3C in an assay as in f , but with incubation with K48-linked ubiquitin (K48-Ub), and probed with antibody to K48-linked ubiquitin. ( i ) Immunoblot analysis of the ubiquitination of EMCV 3C and respiratory syncytial virus NS1 (RSV NS1) in the assay conditions in f . ( j ) Ubiquitination of EMCV 3C by DTX3L, DTX3L-delD3 and DTX3L-RDTXH in the assay conditions in f . Arrowheads ( f – h,j ) indicate ubiquitinated forms. ( k ) Binding of DTX3L-RING-DTXH to EMCV 3C (top) or HRV 3C (bottom), with DTX3L-RING-DTXH concentrations of 10, 5, 2.5, 1.25 and 0.625 μM (for EMCV 3C) or 8.5, 4.25, 2.13, 1.06 and 0.53 μM (for HRV 3C), assessed by real-time biolayer interferometry and presented as sensorgrams (left); solid lines indicate reference-corrected data. Right, orientation of biosensor pin. ( l ) Ubiquitination of EMCV 3C in various HEK293T cells transfected with various constructs (above lanes), assessed by immunoprecipitation of lysates with anti-V5 beads and then with anti-V5 in solution, followed by immunoblot analysis with antibody to hemagglutinin-tagged ubiquitin and anti-V5. * P < 0.05, versus uninfected cells (unpaired t -test). Data are representative of at least three independent experiments (mean and s.e.m. in c ).

Techniques: Immunostaining, Infection, Western Blot, Immunoprecipitation, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Sequencing, Molecular Weight, Purification, Chromatography, Binding Assay, Transfection, Construct

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