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Santa Cruz Biotechnology mouse monoclonal anti human irf3
Neutralization of TLR2 blocks gp120-mediated IFNβ production and <t>IRF3</t> activation. ( a ) Endometrial GECs were pretreated with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibodies (all at 10 μg/ml) before exposure to gp120 (100 ng/ml) or mock treatment (media). FimH and Pam3CSK4 were used as positive controls for activation of TLR4 and TLR2, respectively. Supernatants were collected after 24 h and analyzed by ELISA for IFNβ production. Data shown are mean + s.d. and representative of three separate experiments done on cells isolated from three different tissues. ( b ) Epithelial monolayers were fixed after 2 h of exposure of gp120 with and without pretreatment with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibody and stained for IRF3. Propidium iodide was used to stain nuclei. Images were captures by a laser-scanning confocal microscopy. Magnification × 1260. Images are representative of one of three separate experiments done on cells isolated from three different tissues. ( c ) Quantitation of IRF3 colocalization were done by Image J software and presented in the graph. Significance was calculated by one-way ANOVA and IRF3 colocalization in all treatments were compared with mock treatment. * P
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1) Product Images from "Interferon-β induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier"

Article Title: Interferon-β induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier

Journal: Cellular and Molecular Immunology

doi: 10.1038/cmi.2017.168

Neutralization of TLR2 blocks gp120-mediated IFNβ production and IRF3 activation. ( a ) Endometrial GECs were pretreated with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibodies (all at 10 μg/ml) before exposure to gp120 (100 ng/ml) or mock treatment (media). FimH and Pam3CSK4 were used as positive controls for activation of TLR4 and TLR2, respectively. Supernatants were collected after 24 h and analyzed by ELISA for IFNβ production. Data shown are mean + s.d. and representative of three separate experiments done on cells isolated from three different tissues. ( b ) Epithelial monolayers were fixed after 2 h of exposure of gp120 with and without pretreatment with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibody and stained for IRF3. Propidium iodide was used to stain nuclei. Images were captures by a laser-scanning confocal microscopy. Magnification × 1260. Images are representative of one of three separate experiments done on cells isolated from three different tissues. ( c ) Quantitation of IRF3 colocalization were done by Image J software and presented in the graph. Significance was calculated by one-way ANOVA and IRF3 colocalization in all treatments were compared with mock treatment. * P
Figure Legend Snippet: Neutralization of TLR2 blocks gp120-mediated IFNβ production and IRF3 activation. ( a ) Endometrial GECs were pretreated with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibodies (all at 10 μg/ml) before exposure to gp120 (100 ng/ml) or mock treatment (media). FimH and Pam3CSK4 were used as positive controls for activation of TLR4 and TLR2, respectively. Supernatants were collected after 24 h and analyzed by ELISA for IFNβ production. Data shown are mean + s.d. and representative of three separate experiments done on cells isolated from three different tissues. ( b ) Epithelial monolayers were fixed after 2 h of exposure of gp120 with and without pretreatment with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibody and stained for IRF3. Propidium iodide was used to stain nuclei. Images were captures by a laser-scanning confocal microscopy. Magnification × 1260. Images are representative of one of three separate experiments done on cells isolated from three different tissues. ( c ) Quantitation of IRF3 colocalization were done by Image J software and presented in the graph. Significance was calculated by one-way ANOVA and IRF3 colocalization in all treatments were compared with mock treatment. * P

Techniques Used: Neutralization, Activation Assay, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Confocal Microscopy, Quantitation Assay, Software

Induction of IFNβ in endometrial GECs by HIV-1 gp120 is mediated through IRF3. Endometrial GECs were exposed to medium or poly I:C, HIV-1 (10 5 IU/well) or gp120 (100 ng/ml alone or with anti-gp120 neutralizing antibody) for 1–3 h. Cells were fixed and stained for the IRF3 (green fluorescence). Nuclear counterstaining (red fluorescence) was achieved using PI. Images were captured by a laser-scanning confocal microscopy. ( a ) Representative images are shown at 2 h time point from one of three separate experiments. Magnification × 1260. ( b ) IRF3 translocation and nuclear colocalization was measured by Image J software and presented as relative light units. ( c ) Time kinetics of IRF3 colocalization following treatment of endometrial GECs with medium or poly I:C (positive control), HIV-1 or gp120. ( d , e ) Endometrial GECs were incubated with the IRF3 inhibitor, BX795 (1 μM) for 1 h, before exposure with gp120, HIV-1 or poly I:C (positive control). Supernatants were collected after 24 h and assayed by ELISA. Results showed IFNβ production in apical ( d ) and basolateral supernatants ( e ). ( f ) Endometrial GECs were treated with BX795 for 1 h before gp120 or HIV-1 exposure for 2 h. The cells were fixed and stained for IRF3 and nuclei. Images were captured by laser-scanning confocal microscopy. Magnification: × 1260. ( g ) Colocalization was measured by image J software and represented in a bar diagram. h Endometrial GECs were preincubated with BX795 or media (mock) for 1 h and TERs were measured pretreatment and after 24 h of treatment with mock or HIV-1 to check whether BX795 was affecting HIV-1-mediated barrier disruption. Images are representatives of three separate experiments from cells isolated from three individual tissues. * P
Figure Legend Snippet: Induction of IFNβ in endometrial GECs by HIV-1 gp120 is mediated through IRF3. Endometrial GECs were exposed to medium or poly I:C, HIV-1 (10 5 IU/well) or gp120 (100 ng/ml alone or with anti-gp120 neutralizing antibody) for 1–3 h. Cells were fixed and stained for the IRF3 (green fluorescence). Nuclear counterstaining (red fluorescence) was achieved using PI. Images were captured by a laser-scanning confocal microscopy. ( a ) Representative images are shown at 2 h time point from one of three separate experiments. Magnification × 1260. ( b ) IRF3 translocation and nuclear colocalization was measured by Image J software and presented as relative light units. ( c ) Time kinetics of IRF3 colocalization following treatment of endometrial GECs with medium or poly I:C (positive control), HIV-1 or gp120. ( d , e ) Endometrial GECs were incubated with the IRF3 inhibitor, BX795 (1 μM) for 1 h, before exposure with gp120, HIV-1 or poly I:C (positive control). Supernatants were collected after 24 h and assayed by ELISA. Results showed IFNβ production in apical ( d ) and basolateral supernatants ( e ). ( f ) Endometrial GECs were treated with BX795 for 1 h before gp120 or HIV-1 exposure for 2 h. The cells were fixed and stained for IRF3 and nuclei. Images were captured by laser-scanning confocal microscopy. Magnification: × 1260. ( g ) Colocalization was measured by image J software and represented in a bar diagram. h Endometrial GECs were preincubated with BX795 or media (mock) for 1 h and TERs were measured pretreatment and after 24 h of treatment with mock or HIV-1 to check whether BX795 was affecting HIV-1-mediated barrier disruption. Images are representatives of three separate experiments from cells isolated from three individual tissues. * P

Techniques Used: Staining, Fluorescence, Confocal Microscopy, Translocation Assay, Software, Positive Control, Incubation, Enzyme-linked Immunosorbent Assay, Isolation

2) Product Images from "Importin β1 targeting by hepatitis C virus NS3/ 4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response. Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response"

Article Title: Importin β1 targeting by hepatitis C virus NS3/ 4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response. Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/tra.12480

Microscopy‐based High Content Screening (HCS) of IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. A, Overview of the microscopy‐based gene silencing screen. A549 cells plated in 96‐well plates are transduced with 5 independent lentivirus‐encoding short hairpin RNA (shRNA) per gene (1 shRNA per well) at a multiplicity of infection (MOI) of 10 for 4 days to silence expression of 60 nuclear transport factors. A control shRNA NT is included in each 96‐well plate. Cells are infected with Sendai virus (SeV) for 1, 3, 5, 8 or 10 hours before fixation, permeabilization, Hoechst nuclear labeling and antibody staining of IRF3 or NF‐κB p65 with Alexa Fluor 488 (green). Images of cells are captured in 9 pre‐determined fields for each well using an Operetta HCS Microscope. Images are processed using Harmony software to delimitate the nuclear region and measure the fluorescence intensity of IRF3 or NF‐κB p65 within the nucleus. For each 96‐well plate, a fluorescence cut‐off is set to allow automated discrimination of cells with (green) or without (red) IRF3 or NF‐κB p65 nuclear staining and to calculate the percentage of cells for each shRNA‐mediated gene knockdown. Scale bar is equal to 100 μM. B, Representative time course imaging performed with the control shRNA NT showing the nuclear translocation of IRF3 or NF‐κB p65 over a 10‐hour Sendai virus (SeV) infection (1 representative of 9 field images). Scale bar is equal to 100 μM. C, Graphic representation of the microscopy image‐based analysis showing an increase in the percentage of cells with positive nuclear staining for IRF3 or NF‐κB p65 culminating with ~75% of positive cells at 5 hours post‐infection followed by a decrease to ~30% of positive cells at 10 hours. D, Immunoblot analysis of total cell lysates, cytoplasmic and nuclear extracts of A549 cells infected with lentivirus‐encoding shRNA NT at a MOI of 10 for 3 days and infected with SeV for 0, 1, 3, 5, 8 and 10 hours prior to cell harvesting
Figure Legend Snippet: Microscopy‐based High Content Screening (HCS) of IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. A, Overview of the microscopy‐based gene silencing screen. A549 cells plated in 96‐well plates are transduced with 5 independent lentivirus‐encoding short hairpin RNA (shRNA) per gene (1 shRNA per well) at a multiplicity of infection (MOI) of 10 for 4 days to silence expression of 60 nuclear transport factors. A control shRNA NT is included in each 96‐well plate. Cells are infected with Sendai virus (SeV) for 1, 3, 5, 8 or 10 hours before fixation, permeabilization, Hoechst nuclear labeling and antibody staining of IRF3 or NF‐κB p65 with Alexa Fluor 488 (green). Images of cells are captured in 9 pre‐determined fields for each well using an Operetta HCS Microscope. Images are processed using Harmony software to delimitate the nuclear region and measure the fluorescence intensity of IRF3 or NF‐κB p65 within the nucleus. For each 96‐well plate, a fluorescence cut‐off is set to allow automated discrimination of cells with (green) or without (red) IRF3 or NF‐κB p65 nuclear staining and to calculate the percentage of cells for each shRNA‐mediated gene knockdown. Scale bar is equal to 100 μM. B, Representative time course imaging performed with the control shRNA NT showing the nuclear translocation of IRF3 or NF‐κB p65 over a 10‐hour Sendai virus (SeV) infection (1 representative of 9 field images). Scale bar is equal to 100 μM. C, Graphic representation of the microscopy image‐based analysis showing an increase in the percentage of cells with positive nuclear staining for IRF3 or NF‐κB p65 culminating with ~75% of positive cells at 5 hours post‐infection followed by a decrease to ~30% of positive cells at 10 hours. D, Immunoblot analysis of total cell lysates, cytoplasmic and nuclear extracts of A549 cells infected with lentivirus‐encoding shRNA NT at a MOI of 10 for 3 days and infected with SeV for 0, 1, 3, 5, 8 and 10 hours prior to cell harvesting

Techniques Used: Microscopy, High Content Screening, Translocation Assay, Transduction, shRNA, Infection, Expressing, Labeling, Staining, Software, Fluorescence, Imaging, Cell Harvesting

Effect of silencing importin‐β (IMP‐β) nucleocytoplasmic transport receptor (NTR) and RAN components on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. Importins (IPO4, IPO7, IPO8), transportins (TNPO1/IMPβ2, TNPO2/IPO3, TNPO3/IPO12), as well as proteins involved in protein export (EXP1/XPO1, EXP2/CSE1L, RANBP3), mRNA export (NXT1, NXT2, NXF1, NXF2) and RAN gradient (RAN, NUTF2, RCC1) are silenced in Sendai virus (SeV)‐infected cells. Results are presented as the average of all short hairpin RNAs (shRNAs) in relative percentage of cells containing IRF3 and NF‐κB p65 in the nucleus after normalization of the control shRNA NT to 0 for all time points. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figures S4‐S8
Figure Legend Snippet: Effect of silencing importin‐β (IMP‐β) nucleocytoplasmic transport receptor (NTR) and RAN components on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. Importins (IPO4, IPO7, IPO8), transportins (TNPO1/IMPβ2, TNPO2/IPO3, TNPO3/IPO12), as well as proteins involved in protein export (EXP1/XPO1, EXP2/CSE1L, RANBP3), mRNA export (NXT1, NXT2, NXF1, NXF2) and RAN gradient (RAN, NUTF2, RCC1) are silenced in Sendai virus (SeV)‐infected cells. Results are presented as the average of all short hairpin RNAs (shRNAs) in relative percentage of cells containing IRF3 and NF‐κB p65 in the nucleus after normalization of the control shRNA NT to 0 for all time points. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figures S4‐S8

Techniques Used: Translocation Assay, Infection, shRNA, Activity Assay

Effect of silencing importin (IMP)‐α adaptors on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. IMP‐α adaptors are silenced in Sendai virus (SeV)‐infected cells. Relative percentage of cells containing IRF3 (left) and NF‐κB p65 (right) in the nucleus after normalization of the control short hairpin RNA (shRNA) NT to 0 for all time points. Results are presented as average of all shRNAs for each IMP‐α. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figure S3
Figure Legend Snippet: Effect of silencing importin (IMP)‐α adaptors on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. IMP‐α adaptors are silenced in Sendai virus (SeV)‐infected cells. Relative percentage of cells containing IRF3 (left) and NF‐κB p65 (right) in the nucleus after normalization of the control short hairpin RNA (shRNA) NT to 0 for all time points. Results are presented as average of all shRNAs for each IMP‐α. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figure S3

Techniques Used: Translocation Assay, Infection, shRNA, Activity Assay

Importin β1 (IMPβ1) silencing impairs IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation, IFNB1 induction, and increases Sendai virus (SeV) protein expression and cell apoptosis. A, A total of 3 independent short hairpin RNA (shRNA) targeting IMPβ1 significantly affected nuclear translocation of both IRF3 (left panel) and NF‐κB p65 (middle panel) when compared with the shRNA NT. Relative percentage of cells containing IRF3 and p65 in the nucleus are illustrated after normalization of the control shRNA NT to 0 for all time points. The effect of shRNA‐mediated knockdown on SeV‐induced IFNB1 production is measured in A549 cells stably expressing the firefly luciferase under the control of the IFNB1 promoter (right panel). In addition, the effects of each shRNA on cell proliferation and survival are evaluated using images from the microscopy screen by dividing the total number of nuclei for a given shRNA and dividing it by the total number of nuclei for the shRNA NT control (right panel). B, Immunoblot analysis of A549 cells infected with SeV for 1, 3, 5, 8 or 10 hours following transduction with shRNA NT (control) or shRNA 89 targeting IMPβ1 for 3 days. PARP1 cleavage (arrows) is used as apoptosis readout. C, Human Embryonic Kidney (HEK)293T cells are transfected with 3×FLAG‐NS3/4A expression vector and treated with 1 μM of BILN 2061 PI. At 48 hours post‐transfection, cells are harvested and co‐immunoprecipitation using anti‐FLAG coated beads is performed on cell lysates. NS3 and IMPβ1 interaction is resolved using immunoblot. NS3, NS3/4A precursor, IMPβ1 and cleaved IMPβ1 are resolved using immunoblotting analysis of cell lysates
Figure Legend Snippet: Importin β1 (IMPβ1) silencing impairs IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation, IFNB1 induction, and increases Sendai virus (SeV) protein expression and cell apoptosis. A, A total of 3 independent short hairpin RNA (shRNA) targeting IMPβ1 significantly affected nuclear translocation of both IRF3 (left panel) and NF‐κB p65 (middle panel) when compared with the shRNA NT. Relative percentage of cells containing IRF3 and p65 in the nucleus are illustrated after normalization of the control shRNA NT to 0 for all time points. The effect of shRNA‐mediated knockdown on SeV‐induced IFNB1 production is measured in A549 cells stably expressing the firefly luciferase under the control of the IFNB1 promoter (right panel). In addition, the effects of each shRNA on cell proliferation and survival are evaluated using images from the microscopy screen by dividing the total number of nuclei for a given shRNA and dividing it by the total number of nuclei for the shRNA NT control (right panel). B, Immunoblot analysis of A549 cells infected with SeV for 1, 3, 5, 8 or 10 hours following transduction with shRNA NT (control) or shRNA 89 targeting IMPβ1 for 3 days. PARP1 cleavage (arrows) is used as apoptosis readout. C, Human Embryonic Kidney (HEK)293T cells are transfected with 3×FLAG‐NS3/4A expression vector and treated with 1 μM of BILN 2061 PI. At 48 hours post‐transfection, cells are harvested and co‐immunoprecipitation using anti‐FLAG coated beads is performed on cell lysates. NS3 and IMPβ1 interaction is resolved using immunoblot. NS3, NS3/4A precursor, IMPβ1 and cleaved IMPβ1 are resolved using immunoblotting analysis of cell lysates

Techniques Used: Translocation Assay, Expressing, shRNA, Stable Transfection, Luciferase, Microscopy, Infection, Transduction, Transfection, Plasmid Preparation, Immunoprecipitation

Effect of silencing nucleoporins (Nups) on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. Cytoplasmic FG‐Nups and filaments (RANBP2, NUP214, NUPL2), outer‐ring Nups (NUP43, NUP107, NUP160), linker Nups (NUP93, NUP88) and central FG‐Nups (NUP54, NUP35, NUPL1) are silenced in Sendai virus (SeV)‐infected cells. Results are presented as the average of all short hairpin RNAs (shRNAs) in relative percentage of cells containing IRF3 and NF‐κB p65 in the nucleus after normalization of the control shRNA NT to 0 for all time points. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figures S9‐S12
Figure Legend Snippet: Effect of silencing nucleoporins (Nups) on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. Cytoplasmic FG‐Nups and filaments (RANBP2, NUP214, NUPL2), outer‐ring Nups (NUP43, NUP107, NUP160), linker Nups (NUP93, NUP88) and central FG‐Nups (NUP54, NUP35, NUPL1) are silenced in Sendai virus (SeV)‐infected cells. Results are presented as the average of all short hairpin RNAs (shRNAs) in relative percentage of cells containing IRF3 and NF‐κB p65 in the nucleus after normalization of the control shRNA NT to 0 for all time points. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figures S9‐S12

Techniques Used: Translocation Assay, Infection, shRNA, Activity Assay

NS3/4A‐mediated cleavage of importin β1 (IMPβ1) and interferon‐β (IFNB1) inhibition are completely restored by expression of NS3/4A cleavage‐resistant IMPβ1 variant (IMPβ1 CR ) and treatment with BILN 2061 PI. A, Human Embryonic Kidney (HEK)293T MAVS knockout (KO) cells were transfected with an empty vector or an NS3/4A, MAVS WT , cleavage resistant MAVS C508R and pIFNB1‐LUC expression plasmids, as indicated, for a total of 48 hours. BILN2061 was used as a positive control to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with Sendai virus (SeV) for 16 hours and luciferase signal was resolved. Average pIFNB1 induction folds are from 6 biological replicates. Data were analyzed using 1‐way ANOVA and significance using Bonferroni post hoc test. B, HEK293T MAVS KO cells were transfected with cleavage resistant MAVS C508R , an empty vector or NS3/4A, as indicated, for a total of 48 hours. BILN2061 was used as a positive control to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with SeV for 16 hours and were harvested for western blot analysis. Immunoblots of IMPβ1, MAVS, IFIT1 and Actin are presented to complement luciferase results from (A). C, HEK293T MAVS KO cells reconstituted with cleavage resistant MAVS C508R were transfected with an empty vector or NS3/4A, IMPβ1 WT or cleavage resistant IMPβ1 CR and pIFNB1‐LUC expression plasmids, as indicated, for a total of 48 hours. BILN2061 PI was used to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with SeV for 16 hours and luciferase signal was resolved. Average pIFNB1 induction folds are from 6 biological replicates. Data were analyzed using 1‐way ANOVA and significance using Bonferroni post hoc test. D, A549 cells were transfected with MAVS C508R , an empty vector or NS3/4A, for a total of 48 hours, as indicated. At 40 hours post‐transfection, cells were either uninfected (time 0) or infected with SeV for 2, 4, 6 and 8 hours. Cells were fixed, permeabilized and stained for IRF3 and IMPβ1, and nuclear positive cells were resolved using immunofluorescence. E, A549 cells were transfected with MAVS C508R , an empty vector or NS3/4A, for a total of 48 hours, as indicated. Concurrently, cells are treated with DMSO (vehicle) or BILN2061 PI. At 44 hours post‐transfection, cells were infected with SeV for 4 hours. Cells were fixed, permeabilized and stained for IRF3, and nuclear positive cells were resolved using immunofluorescence. F, A549 cells were transfected with MAVS C508R , an empty vector or cleavage resistant IMPβ1 CR for a total of 48 hours, as indicated. At 44 hours post‐transfection, cells were infected with SeV for 4 hours. Cells were fixed, permeabilized and stained for IRF3, and nuclear positive cells were resolved using immunofluorescence.
Figure Legend Snippet: NS3/4A‐mediated cleavage of importin β1 (IMPβ1) and interferon‐β (IFNB1) inhibition are completely restored by expression of NS3/4A cleavage‐resistant IMPβ1 variant (IMPβ1 CR ) and treatment with BILN 2061 PI. A, Human Embryonic Kidney (HEK)293T MAVS knockout (KO) cells were transfected with an empty vector or an NS3/4A, MAVS WT , cleavage resistant MAVS C508R and pIFNB1‐LUC expression plasmids, as indicated, for a total of 48 hours. BILN2061 was used as a positive control to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with Sendai virus (SeV) for 16 hours and luciferase signal was resolved. Average pIFNB1 induction folds are from 6 biological replicates. Data were analyzed using 1‐way ANOVA and significance using Bonferroni post hoc test. B, HEK293T MAVS KO cells were transfected with cleavage resistant MAVS C508R , an empty vector or NS3/4A, as indicated, for a total of 48 hours. BILN2061 was used as a positive control to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with SeV for 16 hours and were harvested for western blot analysis. Immunoblots of IMPβ1, MAVS, IFIT1 and Actin are presented to complement luciferase results from (A). C, HEK293T MAVS KO cells reconstituted with cleavage resistant MAVS C508R were transfected with an empty vector or NS3/4A, IMPβ1 WT or cleavage resistant IMPβ1 CR and pIFNB1‐LUC expression plasmids, as indicated, for a total of 48 hours. BILN2061 PI was used to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with SeV for 16 hours and luciferase signal was resolved. Average pIFNB1 induction folds are from 6 biological replicates. Data were analyzed using 1‐way ANOVA and significance using Bonferroni post hoc test. D, A549 cells were transfected with MAVS C508R , an empty vector or NS3/4A, for a total of 48 hours, as indicated. At 40 hours post‐transfection, cells were either uninfected (time 0) or infected with SeV for 2, 4, 6 and 8 hours. Cells were fixed, permeabilized and stained for IRF3 and IMPβ1, and nuclear positive cells were resolved using immunofluorescence. E, A549 cells were transfected with MAVS C508R , an empty vector or NS3/4A, for a total of 48 hours, as indicated. Concurrently, cells are treated with DMSO (vehicle) or BILN2061 PI. At 44 hours post‐transfection, cells were infected with SeV for 4 hours. Cells were fixed, permeabilized and stained for IRF3, and nuclear positive cells were resolved using immunofluorescence. F, A549 cells were transfected with MAVS C508R , an empty vector or cleavage resistant IMPβ1 CR for a total of 48 hours, as indicated. At 44 hours post‐transfection, cells were infected with SeV for 4 hours. Cells were fixed, permeabilized and stained for IRF3, and nuclear positive cells were resolved using immunofluorescence.

Techniques Used: Inhibition, Expressing, Variant Assay, Knock-Out, Transfection, Plasmid Preparation, Positive Control, Activity Assay, Infection, Luciferase, Western Blot, Staining, Immunofluorescence

3) Product Images from "Deletion of the Human Cytomegalovirus US17 Gene Increases the Ratio of Genomes per Infectious Unit and Alters Regulation of Immune and Endoplasmic Reticulum Stress Response Genes at Early and Late Times after Infection"

Article Title: Deletion of the Human Cytomegalovirus US17 Gene Increases the Ratio of Genomes per Infectious Unit and Alters Regulation of Immune and Endoplasmic Reticulum Stress Response Genes at Early and Late Times after Infection

Journal: Journal of Virology

doi: 10.1128/JVI.02704-13

ΔUS17 inhibits IRF3 nuclear translocation shortly after infection. HFFs were infected with the indicated virus at an MOI of 3.0 for 3 or 12 h and stained with a monoclonal antibody against IRF3. (A and B) Representative images showing IRF3 and
Figure Legend Snippet: ΔUS17 inhibits IRF3 nuclear translocation shortly after infection. HFFs were infected with the indicated virus at an MOI of 3.0 for 3 or 12 h and stained with a monoclonal antibody against IRF3. (A and B) Representative images showing IRF3 and

Techniques Used: Translocation Assay, Infection, Staining

4) Product Images from "GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors"

Article Title: GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors

Journal: Journal of Virology

doi: 10.1128/JVI.01720-17

SBL induces the IRF3–IFN-β–STAT axis and ISG expression. (A) Macrophages were treated or not with SBL (50 nM) for 2 h, and IFN genes were examined by qPCR. (B) Macrophages were treated with 50 nM SBL for the indicated times, and IFN-β levels in cell-free medium were measured by ELISA. (C and D) Macrophages were treated with SBL (50 nM) for the indicated times (C) or with the indicated concentrations of SBL for 2 h (D). The expression of IRF3, STAT1/3, and ISGF3γp48 was determined by immunoblotting. (E and F) Macrophages were treated with the indicated concentrations of SBL for 6 h (E) or 24 h (F), and the expression of different ISGs was measured. (G and H) Macrophages were treated with the neutralization antibody to IFN-α/β receptor (anti-IFNAR; 10 μg/ml) or control IgG for 1 h and then incubated with SBL for an additional 6 h (G) or anti-IFNAR- or control IgG-treated macrophages were incubated with SBL for 24 h prior to HIV (strain Bal) infection (H). The expression of HIV gag was measured at 72 h postinfection. The data are expressed as means and SD of the results of three independent experiments (*, P
Figure Legend Snippet: SBL induces the IRF3–IFN-β–STAT axis and ISG expression. (A) Macrophages were treated or not with SBL (50 nM) for 2 h, and IFN genes were examined by qPCR. (B) Macrophages were treated with 50 nM SBL for the indicated times, and IFN-β levels in cell-free medium were measured by ELISA. (C and D) Macrophages were treated with SBL (50 nM) for the indicated times (C) or with the indicated concentrations of SBL for 2 h (D). The expression of IRF3, STAT1/3, and ISGF3γp48 was determined by immunoblotting. (E and F) Macrophages were treated with the indicated concentrations of SBL for 6 h (E) or 24 h (F), and the expression of different ISGs was measured. (G and H) Macrophages were treated with the neutralization antibody to IFN-α/β receptor (anti-IFNAR; 10 μg/ml) or control IgG for 1 h and then incubated with SBL for an additional 6 h (G) or anti-IFNAR- or control IgG-treated macrophages were incubated with SBL for 24 h prior to HIV (strain Bal) infection (H). The expression of HIV gag was measured at 72 h postinfection. The data are expressed as means and SD of the results of three independent experiments (*, P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Neutralization, Incubation, Infection

5) Product Images from "Human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: Outcome with influenza A virus is unique"

Article Title: Human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: Outcome with influenza A virus is unique

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.152327499

Activation of IRF-3 by either influenza A virus or influenza B virus is not mediated by IFN-α/β and does not require the synthesis of virus-encoded proteins. ( A ) IRF-3 dimerization assay. A549 cells were either mock-infected (M, lanes 1 and 5), or infected for 4 h with either influenza B virus (B, lane 2) or influenza A virus (A, lane 6). GRE cells were either mock-infected (M, lane 7), or infected for 4 h with influenza A virus (A, lane 8). GRE cells in the presence of CM were either mock-infected (M, lanes 3 and 9), or infected for 4 h with either influenza B virus (B, lane 4) or influenza A virus (A, lane 10). An aliquot of the cell extracts was treated with deoxycholate and subjected to electrophoresis on a 7.5% native gel. The monomer (m) and dimer (d) forms of IRF-3 were identified by immunoblotting with the SL-12 monoclonal antibody. ( B ) Association of CBP with IRF-3 after influenza A or B virus infection. A549 cells were either mock-infected (M, lane 1), or infected for 4 h with either influenza A virus (lane 2) or influenza B virus (B, lane 3). Cell extracts were immunoprecipitated with the SL-12 antibody (anti-IRF-3), and immunoprecipitates were analyzed by Western blots with CBP antibody. Lane 4, cell extract from mock-infected cells was immunoprecipitated with CBP antibody to provide a marker lane for CBP.
Figure Legend Snippet: Activation of IRF-3 by either influenza A virus or influenza B virus is not mediated by IFN-α/β and does not require the synthesis of virus-encoded proteins. ( A ) IRF-3 dimerization assay. A549 cells were either mock-infected (M, lanes 1 and 5), or infected for 4 h with either influenza B virus (B, lane 2) or influenza A virus (A, lane 6). GRE cells were either mock-infected (M, lane 7), or infected for 4 h with influenza A virus (A, lane 8). GRE cells in the presence of CM were either mock-infected (M, lanes 3 and 9), or infected for 4 h with either influenza B virus (B, lane 4) or influenza A virus (A, lane 10). An aliquot of the cell extracts was treated with deoxycholate and subjected to electrophoresis on a 7.5% native gel. The monomer (m) and dimer (d) forms of IRF-3 were identified by immunoblotting with the SL-12 monoclonal antibody. ( B ) Association of CBP with IRF-3 after influenza A or B virus infection. A549 cells were either mock-infected (M, lane 1), or infected for 4 h with either influenza A virus (lane 2) or influenza B virus (B, lane 3). Cell extracts were immunoprecipitated with the SL-12 antibody (anti-IRF-3), and immunoprecipitates were analyzed by Western blots with CBP antibody. Lane 4, cell extract from mock-infected cells was immunoprecipitated with CBP antibody to provide a marker lane for CBP.

Techniques Used: Activation Assay, Infection, Electrophoresis, Immunoprecipitation, Western Blot, Marker

6) Product Images from "Characterization of KIR intermediate promoters reveals four promoter types associated with distinct expression patterns of KIR subtypes"

Article Title: Characterization of KIR intermediate promoters reveals four promoter types associated with distinct expression patterns of KIR subtypes

Journal: Genes and immunity

doi: 10.1038/gene.2015.56

Comparison of the KIR2DL1 ProI region with other KIR genes. ( a ) The 240 bp ProI region of KIR2DL1 is shown, with only nucleotide differences displayed for other KIR genes. The location of the KIR2DL1 sequence relative to the start codon of the gene is indicated in bold at the right end of each line of sequence. Dashes indicate deletions relative to the KIR2DL1 sequence. Underlined bold sequence represents putative transcription factor-binding elements. The single bold A residue denoted by an asterisk indicates the transcription start site determined for KIR2DL1 ProI. KIR2DL1*003 represents the unique sequence found in the KIR2DL1*003 allele, and KIR3DL1*005 represent the sequence found in the KIR3DL1*001, *004 , and *005 alleles. KIR listed in red print are genes with low homology to the KIR2DL1 ProI region. The complete sequence of the KIR3DL3 gene upstream of the Ets-1 site is shown due to a very low level of homology with KIR2DL1 in this region. ( b ) Luciferase activity of pGL3 constructs containing the KIR promoter regions depicted in (a). Constructs were transfected into the cell lines listed, and relative luciferase activity was determined 48 hours post-transfection. Values represent the mean, and error bars indicate the standard deviation of at least 3 independent experiments. KIR genes listed in red type correspond to the divergent KIR gene sequences identified in (a).
Figure Legend Snippet: Comparison of the KIR2DL1 ProI region with other KIR genes. ( a ) The 240 bp ProI region of KIR2DL1 is shown, with only nucleotide differences displayed for other KIR genes. The location of the KIR2DL1 sequence relative to the start codon of the gene is indicated in bold at the right end of each line of sequence. Dashes indicate deletions relative to the KIR2DL1 sequence. Underlined bold sequence represents putative transcription factor-binding elements. The single bold A residue denoted by an asterisk indicates the transcription start site determined for KIR2DL1 ProI. KIR2DL1*003 represents the unique sequence found in the KIR2DL1*003 allele, and KIR3DL1*005 represent the sequence found in the KIR3DL1*001, *004 , and *005 alleles. KIR listed in red print are genes with low homology to the KIR2DL1 ProI region. The complete sequence of the KIR3DL3 gene upstream of the Ets-1 site is shown due to a very low level of homology with KIR2DL1 in this region. ( b ) Luciferase activity of pGL3 constructs containing the KIR promoter regions depicted in (a). Constructs were transfected into the cell lines listed, and relative luciferase activity was determined 48 hours post-transfection. Values represent the mean, and error bars indicate the standard deviation of at least 3 independent experiments. KIR genes listed in red type correspond to the divergent KIR gene sequences identified in (a).

Techniques Used: Sequencing, Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Standard Deviation

7) Product Images from "A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response"

Article Title: A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response

Journal: Journal of Virology

doi: 10.1128/JVI.01175-15

R6 competes with IRF3 for binding to CBP. (A) Telomerized RFs were transfected with pcDNA3.1-R6HA (5, 10, or 20 μg DNA) or empty pcDNA3.1 for 40 h and subsequently transfected with poly(I · C) for 6 h. Nuclear lysates were immunoprecipitated
Figure Legend Snippet: R6 competes with IRF3 for binding to CBP. (A) Telomerized RFs were transfected with pcDNA3.1-R6HA (5, 10, or 20 μg DNA) or empty pcDNA3.1 for 40 h and subsequently transfected with poly(I · C) for 6 h. Nuclear lysates were immunoprecipitated

Techniques Used: Binding Assay, Transfection, Immunoprecipitation

Potential model of IFN inhibition by R6. Upon detection of RRV infection by TLR3, RIG-I, or MDA-5, TBK1 is activated and subsequently phosphorylates IRF3. pIRF3 then dimerizes and translocates to the nucleus. Within the nucleus, R6 binds to the transcriptional
Figure Legend Snippet: Potential model of IFN inhibition by R6. Upon detection of RRV infection by TLR3, RIG-I, or MDA-5, TBK1 is activated and subsequently phosphorylates IRF3. pIRF3 then dimerizes and translocates to the nucleus. Within the nucleus, R6 binds to the transcriptional

Techniques Used: Inhibition, Infection, Multiple Displacement Amplification

8) Product Images from "Zika Virus Non-Structural Protein NS5 Inhibits the RIG-I Pathway and Interferon Lambda 1 Promoter Activation by Targeting IKK Epsilon"

Article Title: Zika Virus Non-Structural Protein NS5 Inhibits the RIG-I Pathway and Interferon Lambda 1 Promoter Activation by Targeting IKK Epsilon

Journal: Viruses

doi: 10.3390/v11111024

ZIKV NS5 inhibits IFN-λ1-promoter activation on both IRF3 and NF-ĸB pathways. HEK293 cells were co-transfected with expression plasmids for ∆RIG-I and ZIKV NS3 or NS5 plasmids, and with IFN-λ1-promoter-luciferase reporter plasmids which were mutated on ( A ) IRF3 or ( B ) NF-ĸB1 binding sites. An expression plasmid for HCV NS3/4A was used as a control. Luc activity was measured at 24 h after transfection. ** indicate a significant reduction in promoter activation ( p
Figure Legend Snippet: ZIKV NS5 inhibits IFN-λ1-promoter activation on both IRF3 and NF-ĸB pathways. HEK293 cells were co-transfected with expression plasmids for ∆RIG-I and ZIKV NS3 or NS5 plasmids, and with IFN-λ1-promoter-luciferase reporter plasmids which were mutated on ( A ) IRF3 or ( B ) NF-ĸB1 binding sites. An expression plasmid for HCV NS3/4A was used as a control. Luc activity was measured at 24 h after transfection. ** indicate a significant reduction in promoter activation ( p

Techniques Used: Activation Assay, Transfection, Expressing, Luciferase, Binding Assay, Plasmid Preparation, Activity Assay

ZIKV NS5 inhibits IFN-λ1-promoter activation on all the components of the RIG-I pathway. HEK293 cells were co-transfected with expression plasmids for ( A ) ∆RIG-I, ( B ) MAVS, ( C ) TBK-1, ( D ) the constitutively active form of IRF3 (IRF3-5D), and ( E , F ) IKKε, and with expression plasmids for ZIKV NS3 and NS5. An expression plasmid for HCV NS3/4A was used as a control. Luc activity was measured at 24 h after transfection. ** indicate a significant reduction in promoter activation ( p
Figure Legend Snippet: ZIKV NS5 inhibits IFN-λ1-promoter activation on all the components of the RIG-I pathway. HEK293 cells were co-transfected with expression plasmids for ( A ) ∆RIG-I, ( B ) MAVS, ( C ) TBK-1, ( D ) the constitutively active form of IRF3 (IRF3-5D), and ( E , F ) IKKε, and with expression plasmids for ZIKV NS3 and NS5. An expression plasmid for HCV NS3/4A was used as a control. Luc activity was measured at 24 h after transfection. ** indicate a significant reduction in promoter activation ( p

Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Activity Assay

ZIKV NS5 inhibits phosphorylation of IRF3 and IKKε. HEK293 cells were co-transfected with expression plasmids for ( A ) ∆RIG-I (400 ng/well) and wtIRF3 (400 ng/well), ( B ) MAVS (400 ng/well), ( C ) TBK-1 (400 ng/well), ( D ) IKKε (40 ng/well and 30 ng/well) and ( E ) the constitutively active form of IRF3 (IRF3-5D) (400 ng/well), and with expression plasmids for ZIKV NS3 and NS5 (40 ng/well and 400 ng/well, for lower panel on IKKε NS5 plasmid amounts were 200 ng/400 ng/800 ng/1600 ng per well). An expression plasmid for HCV NS3/4A was used as a control. Expression levels and the phosphorylation status of the proteins were detected with immunoblotting.
Figure Legend Snippet: ZIKV NS5 inhibits phosphorylation of IRF3 and IKKε. HEK293 cells were co-transfected with expression plasmids for ( A ) ∆RIG-I (400 ng/well) and wtIRF3 (400 ng/well), ( B ) MAVS (400 ng/well), ( C ) TBK-1 (400 ng/well), ( D ) IKKε (40 ng/well and 30 ng/well) and ( E ) the constitutively active form of IRF3 (IRF3-5D) (400 ng/well), and with expression plasmids for ZIKV NS3 and NS5 (40 ng/well and 400 ng/well, for lower panel on IKKε NS5 plasmid amounts were 200 ng/400 ng/800 ng/1600 ng per well). An expression plasmid for HCV NS3/4A was used as a control. Expression levels and the phosphorylation status of the proteins were detected with immunoblotting.

Techniques Used: Transfection, Expressing, Plasmid Preparation

9) Product Images from "Human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: Outcome with influenza A virus is unique"

Article Title: Human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: Outcome with influenza A virus is unique

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.152327499

Activation of IRF-3 by either influenza A virus or influenza B virus is not mediated by IFN-α/β and does not require the synthesis of virus-encoded proteins. ( A ) IRF-3 dimerization assay. A549 cells were either mock-infected (M, lanes 1 and 5), or infected for 4 h with either influenza B virus (B, lane 2) or influenza A virus (A, lane 6). GRE cells were either mock-infected (M, lane 7), or infected for 4 h with influenza A virus (A, lane 8). GRE cells in the presence of CM were either mock-infected (M, lanes 3 and 9), or infected for 4 h with either influenza B virus (B, lane 4) or influenza A virus (A, lane 10). An aliquot of the cell extracts was treated with deoxycholate and subjected to electrophoresis on a 7.5% native gel. The monomer (m) and dimer (d) forms of IRF-3 were identified by immunoblotting with the SL-12 monoclonal antibody. ( B ) Association of CBP with IRF-3 after influenza A or B virus infection. A549 cells were either mock-infected (M, lane 1), or infected for 4 h with either influenza A virus (lane 2) or influenza B virus (B, lane 3). Cell extracts were immunoprecipitated with the SL-12 antibody (anti-IRF-3), and immunoprecipitates were analyzed by Western blots with CBP antibody. Lane 4, cell extract from mock-infected cells was immunoprecipitated with CBP antibody to provide a marker lane for CBP.
Figure Legend Snippet: Activation of IRF-3 by either influenza A virus or influenza B virus is not mediated by IFN-α/β and does not require the synthesis of virus-encoded proteins. ( A ) IRF-3 dimerization assay. A549 cells were either mock-infected (M, lanes 1 and 5), or infected for 4 h with either influenza B virus (B, lane 2) or influenza A virus (A, lane 6). GRE cells were either mock-infected (M, lane 7), or infected for 4 h with influenza A virus (A, lane 8). GRE cells in the presence of CM were either mock-infected (M, lanes 3 and 9), or infected for 4 h with either influenza B virus (B, lane 4) or influenza A virus (A, lane 10). An aliquot of the cell extracts was treated with deoxycholate and subjected to electrophoresis on a 7.5% native gel. The monomer (m) and dimer (d) forms of IRF-3 were identified by immunoblotting with the SL-12 monoclonal antibody. ( B ) Association of CBP with IRF-3 after influenza A or B virus infection. A549 cells were either mock-infected (M, lane 1), or infected for 4 h with either influenza A virus (lane 2) or influenza B virus (B, lane 3). Cell extracts were immunoprecipitated with the SL-12 antibody (anti-IRF-3), and immunoprecipitates were analyzed by Western blots with CBP antibody. Lane 4, cell extract from mock-infected cells was immunoprecipitated with CBP antibody to provide a marker lane for CBP.

Techniques Used: Activation Assay, Infection, Electrophoresis, Immunoprecipitation, Western Blot, Marker

10) Product Images from "Human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: Outcome with influenza A virus is unique"

Article Title: Human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: Outcome with influenza A virus is unique

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.152327499

Influenza B virus, but not influenza A virus, induces the production of mature, functional mRNAs encoded by ISRE-controlled cellular genes. A549 cells were either mock-infected (−), infected with influenza A virus (designated A), or infected with influenza B virus (designated B). Infected cells were collected at the indicated times after infection. ISG15, p56, and 2–5 OAS mRNAs were detected by Northern blot analysis, and the encoded proteins were detected by Western immunoblotting.
Figure Legend Snippet: Influenza B virus, but not influenza A virus, induces the production of mature, functional mRNAs encoded by ISRE-controlled cellular genes. A549 cells were either mock-infected (−), infected with influenza A virus (designated A), or infected with influenza B virus (designated B). Infected cells were collected at the indicated times after infection. ISG15, p56, and 2–5 OAS mRNAs were detected by Northern blot analysis, and the encoded proteins were detected by Western immunoblotting.

Techniques Used: Functional Assay, Infection, Northern Blot, Western Blot

Activation of IRF-3 by either influenza A virus or influenza B virus is not mediated by IFN-α/β and does not require the synthesis of virus-encoded proteins. ( A ) IRF-3 dimerization assay. A549 cells were either mock-infected (M, lanes 1 and 5), or infected for 4 h with either influenza B virus (B, lane 2) or influenza A virus (A, lane 6). GRE cells were either mock-infected (M, lane 7), or infected for 4 h with influenza A virus (A, lane 8). GRE cells in the presence of CM were either mock-infected (M, lanes 3 and 9), or infected for 4 h with either influenza B virus (B, lane 4) or influenza A virus (A, lane 10). An aliquot of the cell extracts was treated with deoxycholate and subjected to electrophoresis on a 7.5% native gel. The monomer (m) and dimer (d) forms of IRF-3 were identified by immunoblotting with the SL-12 monoclonal antibody. ( B ) Association of CBP with IRF-3 after influenza A or B virus infection. A549 cells were either mock-infected (M, lane 1), or infected for 4 h with either influenza A virus (lane 2) or influenza B virus (B, lane 3). Cell extracts were immunoprecipitated with the SL-12 antibody (anti-IRF-3), and immunoprecipitates were analyzed by Western blots with CBP antibody. Lane 4, cell extract from mock-infected cells was immunoprecipitated with CBP antibody to provide a marker lane for CBP.
Figure Legend Snippet: Activation of IRF-3 by either influenza A virus or influenza B virus is not mediated by IFN-α/β and does not require the synthesis of virus-encoded proteins. ( A ) IRF-3 dimerization assay. A549 cells were either mock-infected (M, lanes 1 and 5), or infected for 4 h with either influenza B virus (B, lane 2) or influenza A virus (A, lane 6). GRE cells were either mock-infected (M, lane 7), or infected for 4 h with influenza A virus (A, lane 8). GRE cells in the presence of CM were either mock-infected (M, lanes 3 and 9), or infected for 4 h with either influenza B virus (B, lane 4) or influenza A virus (A, lane 10). An aliquot of the cell extracts was treated with deoxycholate and subjected to electrophoresis on a 7.5% native gel. The monomer (m) and dimer (d) forms of IRF-3 were identified by immunoblotting with the SL-12 monoclonal antibody. ( B ) Association of CBP with IRF-3 after influenza A or B virus infection. A549 cells were either mock-infected (M, lane 1), or infected for 4 h with either influenza A virus (lane 2) or influenza B virus (B, lane 3). Cell extracts were immunoprecipitated with the SL-12 antibody (anti-IRF-3), and immunoprecipitates were analyzed by Western blots with CBP antibody. Lane 4, cell extract from mock-infected cells was immunoprecipitated with CBP antibody to provide a marker lane for CBP.

Techniques Used: Activation Assay, Infection, Electrophoresis, Immunoprecipitation, Western Blot, Marker

11) Product Images from "A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response"

Article Title: A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response

Journal: Journal of Virology

doi: 10.1128/JVI.01175-15

R6 competes with IRF3 for binding to CBP. (A) Telomerized RFs were transfected with pcDNA3.1-R6HA (5, 10, or 20 μg DNA) or empty pcDNA3.1 for 40 h and subsequently transfected with poly(I · C) for 6 h. Nuclear lysates were immunoprecipitated
Figure Legend Snippet: R6 competes with IRF3 for binding to CBP. (A) Telomerized RFs were transfected with pcDNA3.1-R6HA (5, 10, or 20 μg DNA) or empty pcDNA3.1 for 40 h and subsequently transfected with poly(I · C) for 6 h. Nuclear lysates were immunoprecipitated

Techniques Used: Binding Assay, Transfection, Immunoprecipitation

Potential model of IFN inhibition by R6. Upon detection of RRV infection by TLR3, RIG-I, or MDA-5, TBK1 is activated and subsequently phosphorylates IRF3. pIRF3 then dimerizes and translocates to the nucleus. Within the nucleus, R6 binds to the transcriptional
Figure Legend Snippet: Potential model of IFN inhibition by R6. Upon detection of RRV infection by TLR3, RIG-I, or MDA-5, TBK1 is activated and subsequently phosphorylates IRF3. pIRF3 then dimerizes and translocates to the nucleus. Within the nucleus, R6 binds to the transcriptional

Techniques Used: Inhibition, Infection, Multiple Displacement Amplification

Related Articles

Activation Assay:

Article Title: Genome and Infection Characteristics of Human Parechovirus Type 1: The Interplay between Viral Infection and Type I Interferon Antiviral System
Article Snippet: .. Therefore, the activation of IRF3 may be strongly correlated with viral load and also temperature. .. Our other infection model showed the phenomenon of HPeV-dependent IRF3 activation [ ].

other:

Article Title: Hyperosmolarity Invokes Distinct Anti-Inflammatory Mechanisms in Pulmonary Epithelial Cells: Evidence from Signaling and Transcription Layers
Article Snippet: IRF-1 is inhibited by both HOsm conditions for both stimuli.

Article Title: Transcriptional Regulation of PP2A-A? Is Mediated by Multiple Factors Including AP-2?, CREB, ETS-1, and SP-1
Article Snippet: These results suggest that the four cis-elements, CREB, ETS-1, SP-1 and AP-2α in PP2A-Aα gene promoter are likely mediating transcription regulation by the cognate trans -factors in these ocular cell lines.

Article Title: Regulation of the MAD1 promoter by G-CSF
Article Snippet: Antibodies Cytochrom C (Sc-7159, Santa Cruz), Polymerase II (Sc-900, Santa Cruz), AcH3 (#06-866,Upstate), C/EBPα (Sc-61, Santa Cruz), C/EBPβ (Sc-746 and Sc-150, Santa Cruz), C/EBPε (Sc-158, Santa Cruz), STAT3-P (#9131, Cell Signaling), STAT3 (Sc-482, Santa Cruz), actin (A2066, Sigma), HA(3F10) (Boehringer, Mannheim, Germany)

Article Title: Major Human Cytomegalovirus Structural Protein pp65 (ppUL83) Prevents Interferon Response Factor 3 Activation in the Interferon Response
Article Snippet: Thus, pp65 appeared to counteract the hyperphosphorylation of IRF-3 that is associated with nuclear accumulation ( , , ).

Article Title: Major Human Cytomegalovirus Structural Protein pp65 (ppUL83) Prevents Interferon Response Factor 3 Activation in the Interferon Response
Article Snippet: While most pp65 appears to localize to the nucleus following virus entry , we do not yet know whether nuclear or cytoplasmic pp65 is responsible for the impact on IRF-3.

Infection:

Article Title: Genome and Infection Characteristics of Human Parechovirus Type 1: The Interplay between Viral Infection and Type I Interferon Antiviral System
Article Snippet: .. Among the members of IRFs, IRF3 is critically involved in the initial induction of IFNβ when cells are infected by viruses [ ]. .. Although we found that in A549 cells, HPeV1 induced a sustained level of total IRF3 at both 37°C and 39°C, phosphorylated IRF3, the activated form of IRF3, only emerged and lasted until late infection at 37°C, not 39°C.

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  • 92
    Santa Cruz Biotechnology mouse monoclonal anti human irf3
    Neutralization of TLR2 blocks gp120-mediated IFNβ production and <t>IRF3</t> activation. ( a ) Endometrial GECs were pretreated with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibodies (all at 10 μg/ml) before exposure to gp120 (100 ng/ml) or mock treatment (media). FimH and Pam3CSK4 were used as positive controls for activation of TLR4 and TLR2, respectively. Supernatants were collected after 24 h and analyzed by ELISA for IFNβ production. Data shown are mean + s.d. and representative of three separate experiments done on cells isolated from three different tissues. ( b ) Epithelial monolayers were fixed after 2 h of exposure of gp120 with and without pretreatment with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibody and stained for IRF3. Propidium iodide was used to stain nuclei. Images were captures by a laser-scanning confocal microscopy. Magnification × 1260. Images are representative of one of three separate experiments done on cells isolated from three different tissues. ( c ) Quantitation of IRF3 colocalization were done by Image J software and presented in the graph. Significance was calculated by one-way ANOVA and IRF3 colocalization in all treatments were compared with mock treatment. * P
    Mouse Monoclonal Anti Human Irf3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti irf3
    Microscopy‐based High Content Screening (HCS) of IFN regulatory factor 3 <t>(IRF3)</t> and NF‐κB p65 nuclear translocation. A, Overview of the microscopy‐based gene silencing screen. A549 cells plated in 96‐well plates are transduced with 5 independent lentivirus‐encoding short hairpin RNA (shRNA) per gene (1 shRNA per well) at a multiplicity of infection (MOI) of 10 for 4 days to silence expression of 60 nuclear transport factors. A control shRNA NT is included in each 96‐well plate. Cells are infected with Sendai virus (SeV) for 1, 3, 5, 8 or 10 hours before fixation, permeabilization, Hoechst nuclear labeling and antibody staining of IRF3 or NF‐κB p65 with Alexa Fluor 488 (green). Images of cells are captured in 9 pre‐determined fields for each well using an Operetta HCS Microscope. Images are processed using Harmony software to delimitate the nuclear region and measure the fluorescence intensity of IRF3 or NF‐κB p65 within the nucleus. For each 96‐well plate, a fluorescence cut‐off is set to allow automated discrimination of cells with (green) or without (red) IRF3 or NF‐κB p65 nuclear staining and to calculate the percentage of cells for each shRNA‐mediated gene knockdown. Scale bar is equal to 100 μM. B, Representative time course imaging performed with the control shRNA NT showing the nuclear translocation of IRF3 or NF‐κB p65 over a 10‐hour Sendai virus (SeV) infection (1 representative of 9 field images). Scale bar is equal to 100 μM. C, Graphic representation of the microscopy image‐based analysis showing an increase in the percentage of cells with positive nuclear staining for IRF3 or NF‐κB p65 culminating with ~75% of positive cells at 5 hours post‐infection followed by a decrease to ~30% of positive cells at 10 hours. D, Immunoblot analysis of total cell lysates, cytoplasmic and nuclear extracts of A549 cells infected with lentivirus‐encoding shRNA NT at a MOI of 10 for 3 days and infected with SeV for 0, 1, 3, 5, 8 and 10 hours prior to cell harvesting
    Mouse Anti Irf3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology irf3
    ΔUS17 inhibits <t>IRF3</t> nuclear translocation shortly after infection. HFFs were infected with the indicated virus at an MOI of 3.0 for 3 or 12 h and stained with a monoclonal antibody against IRF3. (A and B) Representative images showing IRF3 and
    Irf3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Neutralization of TLR2 blocks gp120-mediated IFNβ production and IRF3 activation. ( a ) Endometrial GECs were pretreated with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibodies (all at 10 μg/ml) before exposure to gp120 (100 ng/ml) or mock treatment (media). FimH and Pam3CSK4 were used as positive controls for activation of TLR4 and TLR2, respectively. Supernatants were collected after 24 h and analyzed by ELISA for IFNβ production. Data shown are mean + s.d. and representative of three separate experiments done on cells isolated from three different tissues. ( b ) Epithelial monolayers were fixed after 2 h of exposure of gp120 with and without pretreatment with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibody and stained for IRF3. Propidium iodide was used to stain nuclei. Images were captures by a laser-scanning confocal microscopy. Magnification × 1260. Images are representative of one of three separate experiments done on cells isolated from three different tissues. ( c ) Quantitation of IRF3 colocalization were done by Image J software and presented in the graph. Significance was calculated by one-way ANOVA and IRF3 colocalization in all treatments were compared with mock treatment. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Interferon-β induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier

    doi: 10.1038/cmi.2017.168

    Figure Lengend Snippet: Neutralization of TLR2 blocks gp120-mediated IFNβ production and IRF3 activation. ( a ) Endometrial GECs were pretreated with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibodies (all at 10 μg/ml) before exposure to gp120 (100 ng/ml) or mock treatment (media). FimH and Pam3CSK4 were used as positive controls for activation of TLR4 and TLR2, respectively. Supernatants were collected after 24 h and analyzed by ELISA for IFNβ production. Data shown are mean + s.d. and representative of three separate experiments done on cells isolated from three different tissues. ( b ) Epithelial monolayers were fixed after 2 h of exposure of gp120 with and without pretreatment with neutralizing antibodies against TLR2, TLR4, TLR5 or isotype control antibody and stained for IRF3. Propidium iodide was used to stain nuclei. Images were captures by a laser-scanning confocal microscopy. Magnification × 1260. Images are representative of one of three separate experiments done on cells isolated from three different tissues. ( c ) Quantitation of IRF3 colocalization were done by Image J software and presented in the graph. Significance was calculated by one-way ANOVA and IRF3 colocalization in all treatments were compared with mock treatment. * P

    Article Snippet: Cells were stained with primary mouse monoclonal anti-human IRF3 (SL-12, Santa Cruz Biotechnology, Inc.) and Alexa Flour 488-conjugated secondary antibody, followed by propidium iodide nuclear counterstaining.

    Techniques: Neutralization, Activation Assay, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Confocal Microscopy, Quantitation Assay, Software

    Induction of IFNβ in endometrial GECs by HIV-1 gp120 is mediated through IRF3. Endometrial GECs were exposed to medium or poly I:C, HIV-1 (10 5 IU/well) or gp120 (100 ng/ml alone or with anti-gp120 neutralizing antibody) for 1–3 h. Cells were fixed and stained for the IRF3 (green fluorescence). Nuclear counterstaining (red fluorescence) was achieved using PI. Images were captured by a laser-scanning confocal microscopy. ( a ) Representative images are shown at 2 h time point from one of three separate experiments. Magnification × 1260. ( b ) IRF3 translocation and nuclear colocalization was measured by Image J software and presented as relative light units. ( c ) Time kinetics of IRF3 colocalization following treatment of endometrial GECs with medium or poly I:C (positive control), HIV-1 or gp120. ( d , e ) Endometrial GECs were incubated with the IRF3 inhibitor, BX795 (1 μM) for 1 h, before exposure with gp120, HIV-1 or poly I:C (positive control). Supernatants were collected after 24 h and assayed by ELISA. Results showed IFNβ production in apical ( d ) and basolateral supernatants ( e ). ( f ) Endometrial GECs were treated with BX795 for 1 h before gp120 or HIV-1 exposure for 2 h. The cells were fixed and stained for IRF3 and nuclei. Images were captured by laser-scanning confocal microscopy. Magnification: × 1260. ( g ) Colocalization was measured by image J software and represented in a bar diagram. h Endometrial GECs were preincubated with BX795 or media (mock) for 1 h and TERs were measured pretreatment and after 24 h of treatment with mock or HIV-1 to check whether BX795 was affecting HIV-1-mediated barrier disruption. Images are representatives of three separate experiments from cells isolated from three individual tissues. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Interferon-β induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier

    doi: 10.1038/cmi.2017.168

    Figure Lengend Snippet: Induction of IFNβ in endometrial GECs by HIV-1 gp120 is mediated through IRF3. Endometrial GECs were exposed to medium or poly I:C, HIV-1 (10 5 IU/well) or gp120 (100 ng/ml alone or with anti-gp120 neutralizing antibody) for 1–3 h. Cells were fixed and stained for the IRF3 (green fluorescence). Nuclear counterstaining (red fluorescence) was achieved using PI. Images were captured by a laser-scanning confocal microscopy. ( a ) Representative images are shown at 2 h time point from one of three separate experiments. Magnification × 1260. ( b ) IRF3 translocation and nuclear colocalization was measured by Image J software and presented as relative light units. ( c ) Time kinetics of IRF3 colocalization following treatment of endometrial GECs with medium or poly I:C (positive control), HIV-1 or gp120. ( d , e ) Endometrial GECs were incubated with the IRF3 inhibitor, BX795 (1 μM) for 1 h, before exposure with gp120, HIV-1 or poly I:C (positive control). Supernatants were collected after 24 h and assayed by ELISA. Results showed IFNβ production in apical ( d ) and basolateral supernatants ( e ). ( f ) Endometrial GECs were treated with BX795 for 1 h before gp120 or HIV-1 exposure for 2 h. The cells were fixed and stained for IRF3 and nuclei. Images were captured by laser-scanning confocal microscopy. Magnification: × 1260. ( g ) Colocalization was measured by image J software and represented in a bar diagram. h Endometrial GECs were preincubated with BX795 or media (mock) for 1 h and TERs were measured pretreatment and after 24 h of treatment with mock or HIV-1 to check whether BX795 was affecting HIV-1-mediated barrier disruption. Images are representatives of three separate experiments from cells isolated from three individual tissues. * P

    Article Snippet: Cells were stained with primary mouse monoclonal anti-human IRF3 (SL-12, Santa Cruz Biotechnology, Inc.) and Alexa Flour 488-conjugated secondary antibody, followed by propidium iodide nuclear counterstaining.

    Techniques: Staining, Fluorescence, Confocal Microscopy, Translocation Assay, Software, Positive Control, Incubation, Enzyme-linked Immunosorbent Assay, Isolation

    Microscopy‐based High Content Screening (HCS) of IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. A, Overview of the microscopy‐based gene silencing screen. A549 cells plated in 96‐well plates are transduced with 5 independent lentivirus‐encoding short hairpin RNA (shRNA) per gene (1 shRNA per well) at a multiplicity of infection (MOI) of 10 for 4 days to silence expression of 60 nuclear transport factors. A control shRNA NT is included in each 96‐well plate. Cells are infected with Sendai virus (SeV) for 1, 3, 5, 8 or 10 hours before fixation, permeabilization, Hoechst nuclear labeling and antibody staining of IRF3 or NF‐κB p65 with Alexa Fluor 488 (green). Images of cells are captured in 9 pre‐determined fields for each well using an Operetta HCS Microscope. Images are processed using Harmony software to delimitate the nuclear region and measure the fluorescence intensity of IRF3 or NF‐κB p65 within the nucleus. For each 96‐well plate, a fluorescence cut‐off is set to allow automated discrimination of cells with (green) or without (red) IRF3 or NF‐κB p65 nuclear staining and to calculate the percentage of cells for each shRNA‐mediated gene knockdown. Scale bar is equal to 100 μM. B, Representative time course imaging performed with the control shRNA NT showing the nuclear translocation of IRF3 or NF‐κB p65 over a 10‐hour Sendai virus (SeV) infection (1 representative of 9 field images). Scale bar is equal to 100 μM. C, Graphic representation of the microscopy image‐based analysis showing an increase in the percentage of cells with positive nuclear staining for IRF3 or NF‐κB p65 culminating with ~75% of positive cells at 5 hours post‐infection followed by a decrease to ~30% of positive cells at 10 hours. D, Immunoblot analysis of total cell lysates, cytoplasmic and nuclear extracts of A549 cells infected with lentivirus‐encoding shRNA NT at a MOI of 10 for 3 days and infected with SeV for 0, 1, 3, 5, 8 and 10 hours prior to cell harvesting

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Importin β1 targeting by hepatitis C virus NS3/ 4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response. Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

    doi: 10.1111/tra.12480

    Figure Lengend Snippet: Microscopy‐based High Content Screening (HCS) of IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. A, Overview of the microscopy‐based gene silencing screen. A549 cells plated in 96‐well plates are transduced with 5 independent lentivirus‐encoding short hairpin RNA (shRNA) per gene (1 shRNA per well) at a multiplicity of infection (MOI) of 10 for 4 days to silence expression of 60 nuclear transport factors. A control shRNA NT is included in each 96‐well plate. Cells are infected with Sendai virus (SeV) for 1, 3, 5, 8 or 10 hours before fixation, permeabilization, Hoechst nuclear labeling and antibody staining of IRF3 or NF‐κB p65 with Alexa Fluor 488 (green). Images of cells are captured in 9 pre‐determined fields for each well using an Operetta HCS Microscope. Images are processed using Harmony software to delimitate the nuclear region and measure the fluorescence intensity of IRF3 or NF‐κB p65 within the nucleus. For each 96‐well plate, a fluorescence cut‐off is set to allow automated discrimination of cells with (green) or without (red) IRF3 or NF‐κB p65 nuclear staining and to calculate the percentage of cells for each shRNA‐mediated gene knockdown. Scale bar is equal to 100 μM. B, Representative time course imaging performed with the control shRNA NT showing the nuclear translocation of IRF3 or NF‐κB p65 over a 10‐hour Sendai virus (SeV) infection (1 representative of 9 field images). Scale bar is equal to 100 μM. C, Graphic representation of the microscopy image‐based analysis showing an increase in the percentage of cells with positive nuclear staining for IRF3 or NF‐κB p65 culminating with ~75% of positive cells at 5 hours post‐infection followed by a decrease to ~30% of positive cells at 10 hours. D, Immunoblot analysis of total cell lysates, cytoplasmic and nuclear extracts of A549 cells infected with lentivirus‐encoding shRNA NT at a MOI of 10 for 3 days and infected with SeV for 0, 1, 3, 5, 8 and 10 hours prior to cell harvesting

    Article Snippet: Following 3 rapid washes, cells were labeled with mouse anti‐IRF3 (SL‐12; Santa Cruz Biotechnology) or rabbit anti‐p65 (C‐20; Santa Cruz Biotechnology) primary antibodies diluted in 5% BSA/0.02% sodium azide/PBS for 2 hours.

    Techniques: Microscopy, High Content Screening, Translocation Assay, Transduction, shRNA, Infection, Expressing, Labeling, Staining, Software, Fluorescence, Imaging, Cell Harvesting

    Effect of silencing importin‐β (IMP‐β) nucleocytoplasmic transport receptor (NTR) and RAN components on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. Importins (IPO4, IPO7, IPO8), transportins (TNPO1/IMPβ2, TNPO2/IPO3, TNPO3/IPO12), as well as proteins involved in protein export (EXP1/XPO1, EXP2/CSE1L, RANBP3), mRNA export (NXT1, NXT2, NXF1, NXF2) and RAN gradient (RAN, NUTF2, RCC1) are silenced in Sendai virus (SeV)‐infected cells. Results are presented as the average of all short hairpin RNAs (shRNAs) in relative percentage of cells containing IRF3 and NF‐κB p65 in the nucleus after normalization of the control shRNA NT to 0 for all time points. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figures S4‐S8

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Importin β1 targeting by hepatitis C virus NS3/ 4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response. Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

    doi: 10.1111/tra.12480

    Figure Lengend Snippet: Effect of silencing importin‐β (IMP‐β) nucleocytoplasmic transport receptor (NTR) and RAN components on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. Importins (IPO4, IPO7, IPO8), transportins (TNPO1/IMPβ2, TNPO2/IPO3, TNPO3/IPO12), as well as proteins involved in protein export (EXP1/XPO1, EXP2/CSE1L, RANBP3), mRNA export (NXT1, NXT2, NXF1, NXF2) and RAN gradient (RAN, NUTF2, RCC1) are silenced in Sendai virus (SeV)‐infected cells. Results are presented as the average of all short hairpin RNAs (shRNAs) in relative percentage of cells containing IRF3 and NF‐κB p65 in the nucleus after normalization of the control shRNA NT to 0 for all time points. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figures S4‐S8

    Article Snippet: Following 3 rapid washes, cells were labeled with mouse anti‐IRF3 (SL‐12; Santa Cruz Biotechnology) or rabbit anti‐p65 (C‐20; Santa Cruz Biotechnology) primary antibodies diluted in 5% BSA/0.02% sodium azide/PBS for 2 hours.

    Techniques: Translocation Assay, Infection, shRNA, Activity Assay

    Effect of silencing importin (IMP)‐α adaptors on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. IMP‐α adaptors are silenced in Sendai virus (SeV)‐infected cells. Relative percentage of cells containing IRF3 (left) and NF‐κB p65 (right) in the nucleus after normalization of the control short hairpin RNA (shRNA) NT to 0 for all time points. Results are presented as average of all shRNAs for each IMP‐α. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figure S3

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Importin β1 targeting by hepatitis C virus NS3/ 4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response. Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

    doi: 10.1111/tra.12480

    Figure Lengend Snippet: Effect of silencing importin (IMP)‐α adaptors on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. IMP‐α adaptors are silenced in Sendai virus (SeV)‐infected cells. Relative percentage of cells containing IRF3 (left) and NF‐κB p65 (right) in the nucleus after normalization of the control short hairpin RNA (shRNA) NT to 0 for all time points. Results are presented as average of all shRNAs for each IMP‐α. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figure S3

    Article Snippet: Following 3 rapid washes, cells were labeled with mouse anti‐IRF3 (SL‐12; Santa Cruz Biotechnology) or rabbit anti‐p65 (C‐20; Santa Cruz Biotechnology) primary antibodies diluted in 5% BSA/0.02% sodium azide/PBS for 2 hours.

    Techniques: Translocation Assay, Infection, shRNA, Activity Assay

    Importin β1 (IMPβ1) silencing impairs IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation, IFNB1 induction, and increases Sendai virus (SeV) protein expression and cell apoptosis. A, A total of 3 independent short hairpin RNA (shRNA) targeting IMPβ1 significantly affected nuclear translocation of both IRF3 (left panel) and NF‐κB p65 (middle panel) when compared with the shRNA NT. Relative percentage of cells containing IRF3 and p65 in the nucleus are illustrated after normalization of the control shRNA NT to 0 for all time points. The effect of shRNA‐mediated knockdown on SeV‐induced IFNB1 production is measured in A549 cells stably expressing the firefly luciferase under the control of the IFNB1 promoter (right panel). In addition, the effects of each shRNA on cell proliferation and survival are evaluated using images from the microscopy screen by dividing the total number of nuclei for a given shRNA and dividing it by the total number of nuclei for the shRNA NT control (right panel). B, Immunoblot analysis of A549 cells infected with SeV for 1, 3, 5, 8 or 10 hours following transduction with shRNA NT (control) or shRNA 89 targeting IMPβ1 for 3 days. PARP1 cleavage (arrows) is used as apoptosis readout. C, Human Embryonic Kidney (HEK)293T cells are transfected with 3×FLAG‐NS3/4A expression vector and treated with 1 μM of BILN 2061 PI. At 48 hours post‐transfection, cells are harvested and co‐immunoprecipitation using anti‐FLAG coated beads is performed on cell lysates. NS3 and IMPβ1 interaction is resolved using immunoblot. NS3, NS3/4A precursor, IMPβ1 and cleaved IMPβ1 are resolved using immunoblotting analysis of cell lysates

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Importin β1 targeting by hepatitis C virus NS3/ 4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response. Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

    doi: 10.1111/tra.12480

    Figure Lengend Snippet: Importin β1 (IMPβ1) silencing impairs IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation, IFNB1 induction, and increases Sendai virus (SeV) protein expression and cell apoptosis. A, A total of 3 independent short hairpin RNA (shRNA) targeting IMPβ1 significantly affected nuclear translocation of both IRF3 (left panel) and NF‐κB p65 (middle panel) when compared with the shRNA NT. Relative percentage of cells containing IRF3 and p65 in the nucleus are illustrated after normalization of the control shRNA NT to 0 for all time points. The effect of shRNA‐mediated knockdown on SeV‐induced IFNB1 production is measured in A549 cells stably expressing the firefly luciferase under the control of the IFNB1 promoter (right panel). In addition, the effects of each shRNA on cell proliferation and survival are evaluated using images from the microscopy screen by dividing the total number of nuclei for a given shRNA and dividing it by the total number of nuclei for the shRNA NT control (right panel). B, Immunoblot analysis of A549 cells infected with SeV for 1, 3, 5, 8 or 10 hours following transduction with shRNA NT (control) or shRNA 89 targeting IMPβ1 for 3 days. PARP1 cleavage (arrows) is used as apoptosis readout. C, Human Embryonic Kidney (HEK)293T cells are transfected with 3×FLAG‐NS3/4A expression vector and treated with 1 μM of BILN 2061 PI. At 48 hours post‐transfection, cells are harvested and co‐immunoprecipitation using anti‐FLAG coated beads is performed on cell lysates. NS3 and IMPβ1 interaction is resolved using immunoblot. NS3, NS3/4A precursor, IMPβ1 and cleaved IMPβ1 are resolved using immunoblotting analysis of cell lysates

    Article Snippet: Following 3 rapid washes, cells were labeled with mouse anti‐IRF3 (SL‐12; Santa Cruz Biotechnology) or rabbit anti‐p65 (C‐20; Santa Cruz Biotechnology) primary antibodies diluted in 5% BSA/0.02% sodium azide/PBS for 2 hours.

    Techniques: Translocation Assay, Expressing, shRNA, Stable Transfection, Luciferase, Microscopy, Infection, Transduction, Transfection, Plasmid Preparation, Immunoprecipitation

    Effect of silencing nucleoporins (Nups) on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. Cytoplasmic FG‐Nups and filaments (RANBP2, NUP214, NUPL2), outer‐ring Nups (NUP43, NUP107, NUP160), linker Nups (NUP93, NUP88) and central FG‐Nups (NUP54, NUP35, NUPL1) are silenced in Sendai virus (SeV)‐infected cells. Results are presented as the average of all short hairpin RNAs (shRNAs) in relative percentage of cells containing IRF3 and NF‐κB p65 in the nucleus after normalization of the control shRNA NT to 0 for all time points. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figures S9‐S12

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Importin β1 targeting by hepatitis C virus NS3/ 4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response. Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

    doi: 10.1111/tra.12480

    Figure Lengend Snippet: Effect of silencing nucleoporins (Nups) on IFN regulatory factor 3 (IRF3) and NF‐κB p65 nuclear translocation. Cytoplasmic FG‐Nups and filaments (RANBP2, NUP214, NUPL2), outer‐ring Nups (NUP43, NUP107, NUP160), linker Nups (NUP93, NUP88) and central FG‐Nups (NUP54, NUP35, NUPL1) are silenced in Sendai virus (SeV)‐infected cells. Results are presented as the average of all short hairpin RNAs (shRNAs) in relative percentage of cells containing IRF3 and NF‐κB p65 in the nucleus after normalization of the control shRNA NT to 0 for all time points. Individual shRNA results on IRF3 and NF‐κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figures S9‐S12

    Article Snippet: Following 3 rapid washes, cells were labeled with mouse anti‐IRF3 (SL‐12; Santa Cruz Biotechnology) or rabbit anti‐p65 (C‐20; Santa Cruz Biotechnology) primary antibodies diluted in 5% BSA/0.02% sodium azide/PBS for 2 hours.

    Techniques: Translocation Assay, Infection, shRNA, Activity Assay

    NS3/4A‐mediated cleavage of importin β1 (IMPβ1) and interferon‐β (IFNB1) inhibition are completely restored by expression of NS3/4A cleavage‐resistant IMPβ1 variant (IMPβ1 CR ) and treatment with BILN 2061 PI. A, Human Embryonic Kidney (HEK)293T MAVS knockout (KO) cells were transfected with an empty vector or an NS3/4A, MAVS WT , cleavage resistant MAVS C508R and pIFNB1‐LUC expression plasmids, as indicated, for a total of 48 hours. BILN2061 was used as a positive control to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with Sendai virus (SeV) for 16 hours and luciferase signal was resolved. Average pIFNB1 induction folds are from 6 biological replicates. Data were analyzed using 1‐way ANOVA and significance using Bonferroni post hoc test. B, HEK293T MAVS KO cells were transfected with cleavage resistant MAVS C508R , an empty vector or NS3/4A, as indicated, for a total of 48 hours. BILN2061 was used as a positive control to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with SeV for 16 hours and were harvested for western blot analysis. Immunoblots of IMPβ1, MAVS, IFIT1 and Actin are presented to complement luciferase results from (A). C, HEK293T MAVS KO cells reconstituted with cleavage resistant MAVS C508R were transfected with an empty vector or NS3/4A, IMPβ1 WT or cleavage resistant IMPβ1 CR and pIFNB1‐LUC expression plasmids, as indicated, for a total of 48 hours. BILN2061 PI was used to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with SeV for 16 hours and luciferase signal was resolved. Average pIFNB1 induction folds are from 6 biological replicates. Data were analyzed using 1‐way ANOVA and significance using Bonferroni post hoc test. D, A549 cells were transfected with MAVS C508R , an empty vector or NS3/4A, for a total of 48 hours, as indicated. At 40 hours post‐transfection, cells were either uninfected (time 0) or infected with SeV for 2, 4, 6 and 8 hours. Cells were fixed, permeabilized and stained for IRF3 and IMPβ1, and nuclear positive cells were resolved using immunofluorescence. E, A549 cells were transfected with MAVS C508R , an empty vector or NS3/4A, for a total of 48 hours, as indicated. Concurrently, cells are treated with DMSO (vehicle) or BILN2061 PI. At 44 hours post‐transfection, cells were infected with SeV for 4 hours. Cells were fixed, permeabilized and stained for IRF3, and nuclear positive cells were resolved using immunofluorescence. F, A549 cells were transfected with MAVS C508R , an empty vector or cleavage resistant IMPβ1 CR for a total of 48 hours, as indicated. At 44 hours post‐transfection, cells were infected with SeV for 4 hours. Cells were fixed, permeabilized and stained for IRF3, and nuclear positive cells were resolved using immunofluorescence.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Importin β1 targeting by hepatitis C virus NS3/ 4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response. Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

    doi: 10.1111/tra.12480

    Figure Lengend Snippet: NS3/4A‐mediated cleavage of importin β1 (IMPβ1) and interferon‐β (IFNB1) inhibition are completely restored by expression of NS3/4A cleavage‐resistant IMPβ1 variant (IMPβ1 CR ) and treatment with BILN 2061 PI. A, Human Embryonic Kidney (HEK)293T MAVS knockout (KO) cells were transfected with an empty vector or an NS3/4A, MAVS WT , cleavage resistant MAVS C508R and pIFNB1‐LUC expression plasmids, as indicated, for a total of 48 hours. BILN2061 was used as a positive control to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with Sendai virus (SeV) for 16 hours and luciferase signal was resolved. Average pIFNB1 induction folds are from 6 biological replicates. Data were analyzed using 1‐way ANOVA and significance using Bonferroni post hoc test. B, HEK293T MAVS KO cells were transfected with cleavage resistant MAVS C508R , an empty vector or NS3/4A, as indicated, for a total of 48 hours. BILN2061 was used as a positive control to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with SeV for 16 hours and were harvested for western blot analysis. Immunoblots of IMPβ1, MAVS, IFIT1 and Actin are presented to complement luciferase results from (A). C, HEK293T MAVS KO cells reconstituted with cleavage resistant MAVS C508R were transfected with an empty vector or NS3/4A, IMPβ1 WT or cleavage resistant IMPβ1 CR and pIFNB1‐LUC expression plasmids, as indicated, for a total of 48 hours. BILN2061 PI was used to inhibit protease activity of NS3/4A protein. At 32 hours post‐transfection, cells were infected with SeV for 16 hours and luciferase signal was resolved. Average pIFNB1 induction folds are from 6 biological replicates. Data were analyzed using 1‐way ANOVA and significance using Bonferroni post hoc test. D, A549 cells were transfected with MAVS C508R , an empty vector or NS3/4A, for a total of 48 hours, as indicated. At 40 hours post‐transfection, cells were either uninfected (time 0) or infected with SeV for 2, 4, 6 and 8 hours. Cells were fixed, permeabilized and stained for IRF3 and IMPβ1, and nuclear positive cells were resolved using immunofluorescence. E, A549 cells were transfected with MAVS C508R , an empty vector or NS3/4A, for a total of 48 hours, as indicated. Concurrently, cells are treated with DMSO (vehicle) or BILN2061 PI. At 44 hours post‐transfection, cells were infected with SeV for 4 hours. Cells were fixed, permeabilized and stained for IRF3, and nuclear positive cells were resolved using immunofluorescence. F, A549 cells were transfected with MAVS C508R , an empty vector or cleavage resistant IMPβ1 CR for a total of 48 hours, as indicated. At 44 hours post‐transfection, cells were infected with SeV for 4 hours. Cells were fixed, permeabilized and stained for IRF3, and nuclear positive cells were resolved using immunofluorescence.

    Article Snippet: Following 3 rapid washes, cells were labeled with mouse anti‐IRF3 (SL‐12; Santa Cruz Biotechnology) or rabbit anti‐p65 (C‐20; Santa Cruz Biotechnology) primary antibodies diluted in 5% BSA/0.02% sodium azide/PBS for 2 hours.

    Techniques: Inhibition, Expressing, Variant Assay, Knock-Out, Transfection, Plasmid Preparation, Positive Control, Activity Assay, Infection, Luciferase, Western Blot, Staining, Immunofluorescence

    SBL induces the IRF3–IFN-β–STAT axis and ISG expression. (A) Macrophages were treated or not with SBL (50 nM) for 2 h, and IFN genes were examined by qPCR. (B) Macrophages were treated with 50 nM SBL for the indicated times, and IFN-β levels in cell-free medium were measured by ELISA. (C and D) Macrophages were treated with SBL (50 nM) for the indicated times (C) or with the indicated concentrations of SBL for 2 h (D). The expression of IRF3, STAT1/3, and ISGF3γp48 was determined by immunoblotting. (E and F) Macrophages were treated with the indicated concentrations of SBL for 6 h (E) or 24 h (F), and the expression of different ISGs was measured. (G and H) Macrophages were treated with the neutralization antibody to IFN-α/β receptor (anti-IFNAR; 10 μg/ml) or control IgG for 1 h and then incubated with SBL for an additional 6 h (G) or anti-IFNAR- or control IgG-treated macrophages were incubated with SBL for 24 h prior to HIV (strain Bal) infection (H). The expression of HIV gag was measured at 72 h postinfection. The data are expressed as means and SD of the results of three independent experiments (*, P

    Journal: Journal of Virology

    Article Title: GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors

    doi: 10.1128/JVI.01720-17

    Figure Lengend Snippet: SBL induces the IRF3–IFN-β–STAT axis and ISG expression. (A) Macrophages were treated or not with SBL (50 nM) for 2 h, and IFN genes were examined by qPCR. (B) Macrophages were treated with 50 nM SBL for the indicated times, and IFN-β levels in cell-free medium were measured by ELISA. (C and D) Macrophages were treated with SBL (50 nM) for the indicated times (C) or with the indicated concentrations of SBL for 2 h (D). The expression of IRF3, STAT1/3, and ISGF3γp48 was determined by immunoblotting. (E and F) Macrophages were treated with the indicated concentrations of SBL for 6 h (E) or 24 h (F), and the expression of different ISGs was measured. (G and H) Macrophages were treated with the neutralization antibody to IFN-α/β receptor (anti-IFNAR; 10 μg/ml) or control IgG for 1 h and then incubated with SBL for an additional 6 h (G) or anti-IFNAR- or control IgG-treated macrophages were incubated with SBL for 24 h prior to HIV (strain Bal) infection (H). The expression of HIV gag was measured at 72 h postinfection. The data are expressed as means and SD of the results of three independent experiments (*, P

    Article Snippet: Mouse anti-IRF3 (SL-12) and goat anti-Mx2 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Neutralization, Incubation, Infection

    ΔUS17 inhibits IRF3 nuclear translocation shortly after infection. HFFs were infected with the indicated virus at an MOI of 3.0 for 3 or 12 h and stained with a monoclonal antibody against IRF3. (A and B) Representative images showing IRF3 and

    Journal: Journal of Virology

    Article Title: Deletion of the Human Cytomegalovirus US17 Gene Increases the Ratio of Genomes per Infectious Unit and Alters Regulation of Immune and Endoplasmic Reticulum Stress Response Genes at Early and Late Times after Infection

    doi: 10.1128/JVI.02704-13

    Figure Lengend Snippet: ΔUS17 inhibits IRF3 nuclear translocation shortly after infection. HFFs were infected with the indicated virus at an MOI of 3.0 for 3 or 12 h and stained with a monoclonal antibody against IRF3. (A and B) Representative images showing IRF3 and

    Article Snippet: Antibody staining was conducted using appropriately diluted primary antibody against pp65 (Fitzgerald Industries, Acton, MA), IRF3 (SL-12; Santa Cruz Biotechnologies, Dallas, TX) and Alexa Fluor 488-conjugated secondary antibody (Life Technologies, Grand Island, NY).

    Techniques: Translocation Assay, Infection, Staining