Journal: Nucleic Acids Research

Article Title: Cumulative contributions of weak DNA determinants to targeting the Drosophila dosage compensation complex

doi: 10.1093/nar/gkm282

Figure Lengend Snippet: A ‘one-hybrid’ transfection assay for isolation of DCC binding sites. ( A ) Summary of the transient transfection assay (inset) and transfected plasmids (not to scale). pGL3-TK is modified by the insertion of candidate DCC binding sites (indicated by question marks) in front of a minimal Herpes Simplex Virus tk promoter, and is transfected together with pRL-TK and either pMSL2-VP16 or pVP16 into Drosophila SL2 cells. ( B ) An example transfection experiment performed with modified pGL3 vectors containing the roX1 c3 cDNA and the roX1 DHS. Numbers above the bars are fold-activation of MSL2-VP16 over VP16 luciferase expression for replicate experiments for the roX1 constructs. Western blots above graphs show expression of activator constructs pVP16, pMSL2-VP16 and pMSL2-eGFP. Transfection of pVP16 resulted in the expression of two proteins migrating at ∼27 kDa. Transfected VP16 and eGFP fusions of MSL2 appear as a single additional band migrating above the endogenous protein.

Article Snippet: Western analysis was performed according to standard laboratory protocols ( ) using antibodies directed against MSL1 , MSL2 and HSV VP16 (Santa Cruz Biotech, SC7545, Santa Cruz, CA, USA).

Techniques: Transfection, Isolation, Binding Assay, Transient Transfection Assay, Modification, Virus, Activation Assay, Luciferase, Expressing, Construct, Western Blot

Journal: Nucleic Acids Research

Article Title: Cumulative contributions of weak DNA determinants to targeting the Drosophila dosage compensation complex

doi: 10.1093/nar/gkm282

Figure Lengend Snippet: Identification of a 40 bp MSL2 binding sequence within DBF12. ( A ) DBF12 is illustrated with the identified DH sites shown as grey bars above a scale in kb. Exons (boxes) and introns (lines) of the Smr gene are indicated, with the arrow showing direction of transcription. Subsequences containing DHS-S are magnified, beneath which the sequences cloned into the firefly luciferase reporter vector pGL3 are indicated. Arrows under the heading ‘orientation’ refer to the direction the fragment was cloned into the pGL3 vector; a forward pointing arrow represents clones with the 3′ end of the sequence closest to the tk promoter. Activation of firefly activity with co-transfected pMSL2-VP16 over firefly luciferase activity with pVP16 is indicated, ±standard deviation. ( B ) Fine mapping deletion and mutant clones derived from DBF12-L15. Mutated bases are underlined. Sequences shown are the reverse complement to their actual insertion in pGL3, to allow easier comparison to GA sequences in DBF6 in Figure 5. Activation of firefly activity with co-transfected pMSL2-VP16 compared to firefly luciferase activity with pVP16 is indicated, ±standard deviation.

Article Snippet: Western analysis was performed according to standard laboratory protocols ( ) using antibodies directed against MSL1 , MSL2 and HSV VP16 (Santa Cruz Biotech, SC7545, Santa Cruz, CA, USA).

Techniques: Binding Assay, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Activation Assay, Activity Assay, Transfection, Standard Deviation, Mutagenesis, Derivative Assay, Comparison

Journal: Nucleic Acids Research

Article Title: Cumulative contributions of weak DNA determinants to targeting the Drosophila dosage compensation complex

doi: 10.1093/nar/gkm282

Figure Lengend Snippet: Restriction of DBF6 with the one-hybrid assay. ( A ) DBF6 is schematized, as in , with two magnified sections. Activation of firefly activity with co-transfected pMSL2-VP16 compared to firefly luciferase activity with pVP16 is indicated, ±standard deviation. ( B ) Fine mapping and deletion clones derived from DBF6-L15.

Article Snippet: Western analysis was performed according to standard laboratory protocols ( ) using antibodies directed against MSL1 , MSL2 and HSV VP16 (Santa Cruz Biotech, SC7545, Santa Cruz, CA, USA).

Techniques: Hybrid Assay, Activation Assay, Activity Assay, Transfection, Luciferase, Standard Deviation, Clone Assay, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Cumulative contributions of weak DNA determinants to targeting the Drosophila dosage compensation complex

doi: 10.1093/nar/gkm282

Figure Lengend Snippet: Restriction of DBF9 with the one-hybrid assay. ( A ) DBF9 is schematized, as in , with two magnified sections. For the illustrated transcripts, filled boxes indicate coding sequence and empty boxes contain untranslated regions. Activation of firefly activity with co-transfected pMSL2-VP16 compared to firefly luciferase activity with pVP16 is indicated, ±standard deviation. ( B ) Fine mapping and deletion clones derived from DBF9-L13.

Article Snippet: Western analysis was performed according to standard laboratory protocols ( ) using antibodies directed against MSL1 , MSL2 and HSV VP16 (Santa Cruz Biotech, SC7545, Santa Cruz, CA, USA).

Techniques: Hybrid Assay, Sequencing, Activation Assay, Activity Assay, Transfection, Luciferase, Standard Deviation, Clone Assay, Derivative Assay

Journal: Journal of Virology

Article Title: Chromatin Assembly on Herpes Simplex Virus 1 DNA Early during a Lytic Infection Is Asf1a Dependent

doi: 10.1128/JVI.01570-12

Figure Lengend Snippet: Asf1a knockdown increased IE viral transcription. HeLa cells were transfected with Asf1a siRNA for 2 days, infected, and harvested at the indicated time. Asf1a siRNA treatment dramatically increased mRNA levels of ICP0 (A) and ICP4 (B), while it did not affect mRNA levels of TK (C) and VP16 (D). Total mRNA was isolated and reverse transcribed. Relative mRNA levels for ICP0, ICP4, TK, and VP16 were determined by qPCR and normalized to 18S rRNA levels. The relative mRNA amount was calculated based on a 3-hpi sample in negative siRNA-treated cells. Three independent experiments were performed, and the error bars represent the standard deviation.

Article Snippet: The following antibodies were used: rabbit monoclonal antibody to Asf1a (Cell Signaling), rabbit polyclonal antibody to tubulin (Santa Cruz), ICP4 (Virusys), and VP16 (Sigma), and mouse monoclonal antibody to CAF-1 (Abcam) and ICP0 (Santa Cruz).

Techniques: Knockdown, Transfection, Infection, Isolation, Reverse Transcription, Standard Deviation

Journal: Journal of Virology

Article Title: Chromatin Assembly on Herpes Simplex Virus 1 DNA Early during a Lytic Infection Is Asf1a Dependent

doi: 10.1128/JVI.01570-12

Figure Lengend Snippet: Asf1a knockdown only increased ICP0 protein expression. Total lysates from siRNA-treated and infected cells at various times postinfection were Western blotted and probed with ICP0, ICP4, and VP16 antibodies. β-Tubulin was used as a loading control.

Article Snippet: The following antibodies were used: rabbit monoclonal antibody to Asf1a (Cell Signaling), rabbit polyclonal antibody to tubulin (Santa Cruz), ICP4 (Virusys), and VP16 (Sigma), and mouse monoclonal antibody to CAF-1 (Abcam) and ICP0 (Santa Cruz).

Techniques: Knockdown, Expressing, Infection, Western Blot, Control

Journal: Journal of Virology

Article Title: Chromatin Assembly on Herpes Simplex Virus 1 DNA Early during a Lytic Infection Is Asf1a Dependent

doi: 10.1128/JVI.01570-12

Figure Lengend Snippet: Asf1a knockdown lowers the deposition of histone H3 on viral promoter regions at 3 hpi. The levels of histone H3 on promoter and transcribed regions of the ICP0 (A), ICP4 (B), TK (C), and VP16 (D) genes were investigated by ChIP. Values are calculated as percentages of input. Student t test values are displayed with asterisks (*, P < 0.01; **, P < 0.001; ***, P < 0.0001). All data are the averages of three independent experiments, and error bars represent the standard deviation.

Article Snippet: The following antibodies were used: rabbit monoclonal antibody to Asf1a (Cell Signaling), rabbit polyclonal antibody to tubulin (Santa Cruz), ICP4 (Virusys), and VP16 (Sigma), and mouse monoclonal antibody to CAF-1 (Abcam) and ICP0 (Santa Cruz).

Techniques: Knockdown, Standard Deviation

Journal: Journal of Virology

Article Title: Chromatin Assembly on Herpes Simplex Virus 1 DNA Early during a Lytic Infection Is Asf1a Dependent

doi: 10.1128/JVI.01570-12

Figure Lengend Snippet: Asf1a knockdown lowers the protection of the viral genome from MNase digestion at 3 hpi. The levels of histone H3 on promoter and transcribed regions of the ICP0 (A), ICP4 (B), TK (C), and VP16 (D) genes were examined by qPCR with 150-bp fragments purified after MNase digestion. Values are calculated as percentages of input. Student t test values are displayed with asterisks (*, P < 0.01; **, P < 0.001; ***, P < 0.0001). All data are the averages of three independent experiments, and error bars represent the standard deviation.

Article Snippet: The following antibodies were used: rabbit monoclonal antibody to Asf1a (Cell Signaling), rabbit polyclonal antibody to tubulin (Santa Cruz), ICP4 (Virusys), and VP16 (Sigma), and mouse monoclonal antibody to CAF-1 (Abcam) and ICP0 (Santa Cruz).

Techniques: Knockdown, Purification, Standard Deviation

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β KO mice exhibit altered responses to HSV-1 skin infection. a Wild type (WT; n = 5 ♀ and 13 ♂) and IL-36β KO mice ( n = 7 ♀ and 12 ♂) were infected with HSV-1 on the flank and skin regions along HSV-1 infected dermatomes collected 6 days post-infection. Viral DNA loads were determined by QPCR. Data are pooled from three independent experiments and shown as means ( ± SD). * p < 0.05 (One-Way ANOVA). b , c Viral ICP4 protein levels were examined by Western blotting ( b ) and quantified using ImageJ software ( c ). GAPDH was used as loading control. Data are representative of three independent experiments and shown as means (±SD) in c (WT: n = 2 female and 4 male; IL-36β KO mice: n = 3 female and 3 male). * p < 0.05 (one-way ANOVA). d Wild type and IL - 36β KO mice were infected with HSV-1 ( n = 3 per group) on the flank and skin RNA isolated 6 days post-infection. Heat-map of genes differentially expressed in the two strains is shown. e Pathway associations of genes identified as differentially expressed in d . f Expression of Oas1 , Eif2ak2, Isg15, Ifitm3, Ifitm2, and Ifit3 mRNAs were examined in wild type and IL-36β KO HSV-1 infected skin ( a ). The mRNAs were normalized against GAPDH and are shown as relative expression compared to female wild-type mice (means ± SD). * p < 0.05; ** p < 0.005 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Mouse monoclonal antibodies to HSV-1 ICP4 (H943, Catalog # sc-69809, RRID:AB_844234, used at 1:500 dilution), IFN-α/βRα Antibody (H-11, Catalog # sc-7391, RRID:AB_2122749, used at 1:800 dilution), IFN-α/βRβ Antibody (F-7, Catalog # sc-137209, RRID:AB_2122750, used at 1:500 dilution) and Mx1 Antibody (E-8, Catalog # sc-398564, RRID:AB_1146318, used at 1:600 dilution) were acquired from Santa Cruz Biotechnology.

Techniques: Infection, Western Blot, Software, Control, Isolation, Expressing

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β induces STAT1- and STAT2-dependent antiviral immunity in keratinocytes. a Human keratinocytes were pre-treated with medium only or IL-36β before infection with HSV-1 (MOI = 0.01). Levels of HSV-1 ICP4 protein were determined by Western blotting and ImageJ analyses using GAPDH as control. b Mouse primary keratinocytes were pre-treated with medium only or IL-36β, followed by HSV-1 infection (MOI = 0.01), and ICP4 levels examined by western blotting. c Wild type ( + / + ) and IL-36β KO (−/−) mouse primary keratinocytes were infected with 0.01 MOI HSV-1 and ICP4 examined by western blotting. d Wild type and STAT1 −/− primary male mouse keratinocytes were treated with medium only or IL-36β followed by HSV-1 infection (MOI = 0.01) for 24 h. Levels of HSV-1 ICP4 and host Mx1 were examined by western blotting. e Wild type and STAT2 −/− primary male mouse keratinocytes were examined after IL-36β pre-treatment and HSV-1 infection using western blotting. a – e Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group); ** p < 0.01. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Mouse monoclonal antibodies to HSV-1 ICP4 (H943, Catalog # sc-69809, RRID:AB_844234, used at 1:500 dilution), IFN-α/βRα Antibody (H-11, Catalog # sc-7391, RRID:AB_2122749, used at 1:800 dilution), IFN-α/βRβ Antibody (F-7, Catalog # sc-137209, RRID:AB_2122750, used at 1:500 dilution) and Mx1 Antibody (E-8, Catalog # sc-398564, RRID:AB_1146318, used at 1:600 dilution) were acquired from Santa Cruz Biotechnology.

Techniques: Infection, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IRF1 is partially involved in immunity against HSV-1. a – e C57BL/6 J (WT) and IRF1 KO mice were infected with HSV-1 on the flank. a Survival was monitored for 16 days. b Weight was measured for 9 days. c Gastrointestinal dysfunction was examined at day 9 post-infection. Red arrow points to disease affected stomach. d Skin lesion sizes were measured (male mice; WT: n = 5; KO: n = 4). e HSV-1 DNA copy numbers in the skin were determined 6 days post-infection (male mice; WT: n = 7; KO: n = 6). f Mouse primary keratinocytes from wild type (WT) and IRF1 KO mice were sequentially treated with medium only or IL-36β as indicated and infected with HSV-1 ( n = 2 biologically independent samples per treatment group). Levels of ICP4 and Mx1 were evaluated by western blotting and ImageJ analyses. g Levels of ICP4 and Mx1 protein were evaluated by western blotting and ImageJ analyses following IL-36β treatment and HSV-1 infection of human control (Ctrl) and IRF1 KO keratinocytes ( n = 2 biologically independent samples per treatment group). a , c * p < 0.05; ** p < 0.01 (Mantel-Cox and Gehan-Breslow-Wilcoxon tests). d – g Quantitative data are shown as means ± SD. * p < 0.05; # p > 0.05 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Mouse monoclonal antibodies to HSV-1 ICP4 (H943, Catalog # sc-69809, RRID:AB_844234, used at 1:500 dilution), IFN-α/βRα Antibody (H-11, Catalog # sc-7391, RRID:AB_2122749, used at 1:800 dilution), IFN-α/βRβ Antibody (F-7, Catalog # sc-137209, RRID:AB_2122750, used at 1:500 dilution) and Mx1 Antibody (E-8, Catalog # sc-398564, RRID:AB_1146318, used at 1:600 dilution) were acquired from Santa Cruz Biotechnology.

Techniques: Infection, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β induced antiviral state is dependent upon IFNAR. a Human keratinocytes were transfected with control (Ctrl) or IFNAR1 gRNA/Cas9 expression plasmids, treated with IL-36β and infected with HSV-1. b Control (Ctrl) or IFNAR2 gRNA/Cas9 expression plasmid transfected human keratinocytes were treated with IL-36β and infected with HSV-1. a , b Levels of ICP4, Mx1, IFNAR1, IFNAR2, and GAPDH were determined using western blotting and ImageJ analyses. c Mouse primary keratinocytes were treated with IL-36β as indicated, incubated with neutralizing antibodies against IFNAR or isotype matched Ig and infected with HSV-1. Levels of ICP4, Mx1 and GAPDH were determined using western blotting and ImageJ analyses. a – c Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Mouse monoclonal antibodies to HSV-1 ICP4 (H943, Catalog # sc-69809, RRID:AB_844234, used at 1:500 dilution), IFN-α/βRα Antibody (H-11, Catalog # sc-7391, RRID:AB_2122749, used at 1:800 dilution), IFN-α/βRβ Antibody (F-7, Catalog # sc-137209, RRID:AB_2122750, used at 1:500 dilution) and Mx1 Antibody (E-8, Catalog # sc-398564, RRID:AB_1146318, used at 1:600 dilution) were acquired from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Expressing, Infection, Plasmid Preparation, Western Blot, Incubation