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rabbit anti ripk3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti ripk3
    Rabbit Anti Ripk3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+ripk3/RIP3+XP+Rabbit+mAb/pm41932973-83-0-16
    Average 95 stars, based on 57 article reviews
    rabbit anti ripk3 - by Bioz Stars, 2026-09
    95/100 stars

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    Related Articles

    Western Blot:

    Article Title: RIPK3 promotes skin inflammation by enhancing IL-36α signaling and necroptosis in keratinocytes
    Article Snippet: The following day after incubation with a proportionate dilution of the HRP-conjugated antibody, the proteins were imaged using the ChemiDoc TM MP Imaging System (Bio-Rad). .. The following antibodies were used for Western blot analysis: anti-RIPK1 (CST, 3493), anti-p-RIPK1 (phospho-Ser166) (CST, 31122), anti-RIPK3 (CST, 95702), anti-p-RIPK3 (phospho-S232) (Abcam, ab195117), anti-MLKL (CST, 37705), anti-p-MLKL (phospho-S345) (Abcam, ab150115), anti-Occludin (Abcam, ab216327), anti-Claudin4 (Abcam, ab15104), anti-Claudin1 (Abcam, ab15098), anti-p-RIPK1 (phospho-Ser166) (CST, 65746), anti-p-RIPK3 (phospho-Ser227) (CST, 93654), anti RIPK3 (CST, 10188), anti-MLKL (Abcam, ab243142), anti-p-MLKL (phospho-S358) (Abcam, ab187091), anti-IL-36 alpha (R&D), anti-MyD88 (CST, 4283), anti-NF-κB (CST, 4764), anti- p-NF-κB (Phospho-Ser536) (CST, 3033), anti-IκBα (CST, 4812) and anti-p-IκBα (Phospho-Ser32/36) (CST, 9246). .. Data analysis was conducted using GraphPad Prism 9.0 software (San Diego, CA, USA).

    Article Title: MLKL depletion enhances chemotherapy-induced apoptosis in colorectal cancer by prolonged retention of TNFR-I in endosomes.
    Article Snippet: 1 Graduate Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Center of Excellence for Cancer and Inflammation, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 4 Chulalongkorn University Laboratory Animal Center, Chulalongkorn University, Bangkok 10330, Thailand 5 Department of Pathology, Tohoku University School of Medicine, Sendai 980-8575, Miyagi, Japan 6 Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Abstract 5-Fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents for various cancers, including cholangiocarcinoma (CCA) and colorectal cancer (CRC).. However, its therapeutic efficiency has remained unsatisfactory.. A better understanding of the molecular mechanisms underlying 5-FU responsiveness is therefore crucial for developing more effective treatment strategies and improving patient survival.

    Virus:

    Article Title: The NSP5, ORF6 and NSP13 of SARS‐CoV‐2 Cooperate to Modulate Inflammatory Cell Death Activation
    Article Snippet: Then, secondary antibodies with HRP were incubated at room temperature for 1 h. Images were acquired via a GE Amersham ImageQuant 800. .. The antibodies used were: anti‐caspase‐8 (Enzo, ALX‐804‐242‐C100, 1:1000), anti‐caspase‐8 (AdipoGen, AG‐20T‐0138‐C100, 1:1000), anti‐cleaved caspase‐8 (CST, 8592, 1:1000), anti‐caspase‐3 (CST, 9662S, 1:1000), anti‐cleaved caspase‐3 (CST, 9661, 1:1000), anti‐caspase‐7 (CST, 9492, 1:1000), anti‐cleaved caspase‐7 (CST, 9491, 1:1000), anti‐caspase‐1 (R&D, mab6215, 1:1000), anti‐caspase‐1 (CST, 2225, 1:1000), anti‐hCoV‐OC43 nucleoprotein (SinoBiological, 40643‐T62, 1:1000), anti‐GSDMD (Abclonal, A20197, 1:1000), anti‐cleaved GSDMD (N terminal) (Abclonal, A22523, 1:1000), anti‐GSDME (Abcam, ab215191, 1:1000), anti‐cleaved IL‐1β (Abclonal, A1112, 1:1000), anti‐pro‐IL‐1β (SinoBiological, 10139‐M201, 1:1000), anti‐SARS‐CoV‐2 nucleoprotein (Abclonal, A18797, 1:1000), anti‐SARS‐CoV‐2 NSP5 (Abclonal, A20198, 1:1000), anti‐NLRP3 (AdipoGen, AG‐20B‐0014, 1:1000), anti‐ZBP1 (CST, 60968S, 1:1000), anti‐RIPK1 (CST, 3493, 1:1000), anti‐RIPK3 (CST, 10188, 1:1000), anti‐pMLKL (abcam, ab187091, 1:1000; CST, 91689S, 1:1000), anti‐MLKL (CST, 14993S, 1:1000), anti‐ASC (AdipoGen, AG‐25B‐0006‐C100, 1:1000), anti‐ACE2 (SinoBiological, 10108‐R003, 1:1000), anti‐TMPRSS2 (SinoBiological, 204314‐T08, 1:1000), anti‐influenza A virus NS1 (Santa Cruz, sc‐130568, 1:1000), anti‐Flag (Sigma, F1804, 1:5000), anti‐HA (Thermo Fisher Scientific, 206183, 1:1000), anti‐Strep (MBL, M211‐3, 1:1000), anti‐GFP (SinoBiological, 13105‐R208, 1:1000), anti‐GST (OriGene, TA150102, 1:1000), anti‐Myc (SinoBiological, 100029‐MM08, 1:1000), anti‐His (OriGene, TA150088, 1:1000), anti‐actin (Proteintech, 66009‐1‐IG, 1:5000), and HRP‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, anti‐rabbit [111‐035‐047], 1:5000; anti‐mouse [315‐035‐047], 1:5000). ..

    Article Title: The NSP5, ORF6 and NSP13 of SARS-CoV-2 Cooperate to Modulate Inflammatory Cell Death Activation.
    Article Snippet: .. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 1:1000), anti-cleaved caspase-3 (CST, 9661, 1:1000), anti-caspase-7 (CST, 9492, 1:1000), anti-cleaved caspase-7 (CST, 9491, 1:1000), anti-caspase-1 (R&D, mab6215, 1:1000), anti-caspase-1 (CST, 2225, 1:1000), anti-hCoVOC43 nucleoprotein (SinoBiological, 40643-T62, 1:1000), anti-GSDMD (Abclonal, A20197, 1:1000), anti-cleaved GSDMD (N terminal) (Abclonal, A22523, 1:1000), anti-GSDME (Abcam, ab215191, 1:1000), anticleaved IL-1β (Abclonal, A1112, 1:1000), anti-pro-IL-1β (SinoBiological, 10139-M201, 1:1000), anti-SARS-CoV-2 nucleoprotein (Abclonal, A18797, 1:1000), anti-SARS-CoV-2 NSP5 (Abclonal, A20198, 1:1000), anti-NLRP3 (AdipoGen, AG-20B-0014, 1:1000), anti-ZBP1 (CST, 60968S, 1:1000), anti-RIPK1 (CST, 3493, 1:1000), anti-RIPK3 (CST, 10188, 1:1000), antipMLKL (abcam, ab187091, 1:1000; CST, 91689S, 1:1000), anti-MLKL (CST, 14993S, 1:1000), anti-ASC (AdipoGen, AG-25B-0006-C100, 1:1000), antiACE2 (SinoBiological, 10108-R003, 1:1000), anti-TMPRSS2 (SinoBiological, 204314-T08, 1:1000), anti-influenza A virus NS1 (Santa Cruz, sc130568, 1:1000), anti-Flag (Sigma, F1804, 1:5000), anti-HA (Thermo Fisher Scientific, 206183, 1:1000), anti-Strep (MBL, M211-3, 1:1000), antiGFP (SinoBiological, 13105-R208, 1:1000), anti-GST (OriGene, TA150102, 1:1000), anti-Myc (SinoBiological, 100029-MM08, 1:1000), anti-His (OriGene, TA150088, 1:1000), anti-actin (Proteintech, 66009-1-IG, 1:5000), and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, anti-rabbit [111-035-047], 1:5000; anti-mouse [315-035-047], 1:5000). ..

    Incubation:

    Article Title: Interleukin-6-Associated Pyroptosis, Apoptosis, and Necroptosis via JAKs/STAT3-RIPK1 Axis: A Potential Mechanism for CD4+ T-Cell Depletion in Bacterial Sepsis
    Article Snippet: .. CD4+ T cells were fixed with 4% paraformaldehyde, permeabilized with 0.03% Triton X-100 for 15 minutes, and incubated overnight at 4°C with primary antibodies: anti-Caspase-8 (AB25901, Abcam, Cambridge, United Kingdom), anti-RIPK3 (No. 10188, Cell Signaling Technology [CST], Danvers, MA), and anti-apoptosisassociated speck-like protein containing a CARD (anti-ASC, No. 78000, CST). .. After washing with phosphate-buffered saline (PBS), cells were incubated with horseradish peroxidase (HRP)-conjugated goat antirabbit immunoglobulin G (1:200, GB23303, Servicebio) for 50 minutes at room temperature, followed by nuclear staining with 4', 6-diamidino-2-phenylindole (DAPI, G1012, Servicebio) for 15 minutes.

    Article Title: Targeting RIG-I alleviates renal tubular epithelial cells PANoptosis during post-traumatic rhabdomyolysis.
    Article Snippet: This is a PDF of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability.. This version will undergo additional copyediting, typesetting and review before it is published in its final form.. As such, this version is no longer the Accepted Manuscript, but it is not yet the definitive Version of Record; we are providing this early version to give early visibility of the article.

    Article Title: GDF15 regulates necroptotic cell death through direct interaction with RIPK3
    Article Snippet: Proteins were then separated on a 12% Bis-Tris SDS-PAGE gel (Bio-Rad) and electroblotted onto PVDF membranes. .. The membranes were blocked with 5% non-fat milk (Nutricia, Zoetermeer, NL) in TBS 1X supplemented with 0.1% Tween 20 (TBS-T) and incubated overnight at 4°C with the following primary antibodies: anti-b-actin (1:1000, 4970S, Cell Signaling, Danvers, MA, USA), anti-GDF15 (1:1000, 79996S, Cell Signaling, Danvers, MA, USA), anti-MLKL (1:1000, 14993S, Cell Signaling, Danvers, MA, USA), anti-PMLKL (1:1000, 91689T, Cell Signaling, Danvers, MA, USA), anti-PRIPK1 (1:1000, 44590S, Cell Signaling, Danvers, MA, USA), anti-PRIPK3 (1:1000, 93654S, Cell Signaling, Danvers, MA, USA), anti-RIPK1 (1:1000, 3493T, Cell Signaling, Danvers, MA, USA), anti-RIPK3 (1:1000, 86671S, Cell Signaling, Danvers, MA, USA), anti-SEMG1 (1:1000, Abbexa, Cambridge, UK), anti-SEMG2 (0.1ug/mL, Abbexa, Cambridge, UK). .. As a secondary antibody, anti-rabbit IgG, HRP linked (1:1000, 7074S, Cell Signaling, Danvers, MA, USA) was used.

    Binding Assay:

    Article Title: Targeting RIG-I alleviates renal tubular epithelial cells PANoptosis during post-traumatic rhabdomyolysis.
    Article Snippet: This is a PDF of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability.. This version will undergo additional copyediting, typesetting and review before it is published in its final form.. As such, this version is no longer the Accepted Manuscript, but it is not yet the definitive Version of Record; we are providing this early version to give early visibility of the article.

    Membrane:

    Article Title: Targeting RIG-I alleviates renal tubular epithelial cells PANoptosis during post-traumatic rhabdomyolysis.
    Article Snippet: This is a PDF of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability.. This version will undergo additional copyediting, typesetting and review before it is published in its final form.. As such, this version is no longer the Accepted Manuscript, but it is not yet the definitive Version of Record; we are providing this early version to give early visibility of the article.

    Flow Cytometry:

    Article Title: MLKL depletion enhances chemotherapy-induced apoptosis in colorectal cancer by prolonged retention of TNFR-I in endosomes.
    Article Snippet: 1 Graduate Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Center of Excellence for Cancer and Inflammation, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 4 Chulalongkorn University Laboratory Animal Center, Chulalongkorn University, Bangkok 10330, Thailand 5 Department of Pathology, Tohoku University School of Medicine, Sendai 980-8575, Miyagi, Japan 6 Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Abstract 5-Fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents for various cancers, including cholangiocarcinoma (CCA) and colorectal cancer (CRC).. However, its therapeutic efficiency has remained unsatisfactory.. A better understanding of the molecular mechanisms underlying 5-FU responsiveness is therefore crucial for developing more effective treatment strategies and improving patient survival.

    Immunofluorescence:

    Article Title: MLKL depletion enhances chemotherapy-induced apoptosis in colorectal cancer by prolonged retention of TNFR-I in endosomes.
    Article Snippet: 1 Graduate Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Center of Excellence for Cancer and Inflammation, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 4 Chulalongkorn University Laboratory Animal Center, Chulalongkorn University, Bangkok 10330, Thailand 5 Department of Pathology, Tohoku University School of Medicine, Sendai 980-8575, Miyagi, Japan 6 Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Abstract 5-Fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents for various cancers, including cholangiocarcinoma (CCA) and colorectal cancer (CRC).. However, its therapeutic efficiency has remained unsatisfactory.. A better understanding of the molecular mechanisms underlying 5-FU responsiveness is therefore crucial for developing more effective treatment strategies and improving patient survival.

    Staining:

    Article Title: MLKL depletion enhances chemotherapy-induced apoptosis in colorectal cancer by prolonged retention of TNFR-I in endosomes.
    Article Snippet: 1 Graduate Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Center of Excellence for Cancer and Inflammation, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 4 Chulalongkorn University Laboratory Animal Center, Chulalongkorn University, Bangkok 10330, Thailand 5 Department of Pathology, Tohoku University School of Medicine, Sendai 980-8575, Miyagi, Japan 6 Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Abstract 5-Fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents for various cancers, including cholangiocarcinoma (CCA) and colorectal cancer (CRC).. However, its therapeutic efficiency has remained unsatisfactory.. A better understanding of the molecular mechanisms underlying 5-FU responsiveness is therefore crucial for developing more effective treatment strategies and improving patient survival.



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    Cell Signaling Technology Inc ripk3
    TBK1 and NF-κB positively regulate IRG1 levels in HMPV-infected human macrophages. MDMs were incubated with 5 or 10 μM of the TBK1 inhibitor BX795 for 30 min before infection with HMPV for 24 h. ( A ) IRG1 and IFN-β mRNA levels ( n ≥ 3) were analysed by qRT-PCR (left panels), while in panel ( B ) IRG1 and GAPDH protein levels were determined by immunoblotting, quantified, and presented with SD relative to uninfected cells treated with DMSO ( n = 2; right panel). (C–K) MDMs were transfected with siRNAs targeting the NF-κB subunit RELA (C–E), IRF1 (F–H), <t>RIPK3</t> (I–K), or control siRNA (siNTC) before infection with HMPV for 6, 9, or 24 h and analysis of IRG1 mRNA levels by qRT-PCR or IRG1, RelA/p65, IRF1, RIPK3, or GAPDH protein levels by immunoblotting. IRG1 mRNA levels were assessed relative to siNTC-transfected, uninfected MDMs for all experiments. ( C, E ) IRG1 mRNA after 6 h ( n = 3) or 24 h HMPV ( n = 3). ( D ) Protein levels of IRG1, RelA/p65, and GAPDH ( n = 3) after 9 h HMPV. ( F, H ) IRG1 mRNA after 6 h ( n = 4) or 24 h of infection ( n = 3). ( G ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( I, K ) IRG1 mRNA after 6 h ( n = 3) or 24 h of infection ( n = 3). ( J ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( L ) MDMs were incubated with 100 nM of the RIPK3 inhibitor GSK 872 for 1 h before infection with HMPV for 24 h. IRG1 mRNA levels were normalized relative to uninfected cells treated with DMSO ( n = 2). Single comparison between control (NTC) siRNA and target-siRNA conditions were calculated using paired t -test with Tukey post-hoc test. * P < .05, ** P < .01, *** P < .001, **** P < .0001; ns = non-significant. See also
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    TBK1 and NF-κB positively regulate IRG1 levels in HMPV-infected human macrophages. MDMs were incubated with 5 or 10 μM of the TBK1 inhibitor BX795 for 30 min before infection with HMPV for 24 h. ( A ) IRG1 and IFN-β mRNA levels ( n ≥ 3) were analysed by qRT-PCR (left panels), while in panel ( B ) IRG1 and GAPDH protein levels were determined by immunoblotting, quantified, and presented with SD relative to uninfected cells treated with DMSO ( n = 2; right panel). (C–K) MDMs were transfected with siRNAs targeting the NF-κB subunit RELA (C–E), IRF1 (F–H), <t>RIPK3</t> (I–K), or control siRNA (siNTC) before infection with HMPV for 6, 9, or 24 h and analysis of IRG1 mRNA levels by qRT-PCR or IRG1, RelA/p65, IRF1, RIPK3, or GAPDH protein levels by immunoblotting. IRG1 mRNA levels were assessed relative to siNTC-transfected, uninfected MDMs for all experiments. ( C, E ) IRG1 mRNA after 6 h ( n = 3) or 24 h HMPV ( n = 3). ( D ) Protein levels of IRG1, RelA/p65, and GAPDH ( n = 3) after 9 h HMPV. ( F, H ) IRG1 mRNA after 6 h ( n = 4) or 24 h of infection ( n = 3). ( G ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( I, K ) IRG1 mRNA after 6 h ( n = 3) or 24 h of infection ( n = 3). ( J ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( L ) MDMs were incubated with 100 nM of the RIPK3 inhibitor GSK 872 for 1 h before infection with HMPV for 24 h. IRG1 mRNA levels were normalized relative to uninfected cells treated with DMSO ( n = 2). Single comparison between control (NTC) siRNA and target-siRNA conditions were calculated using paired t -test with Tukey post-hoc test. * P < .05, ** P < .01, *** P < .001, **** P < .0001; ns = non-significant. See also
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    Image Search Results


    QUE attenuated necroptosis through Nrf2/PERK under LPS exposure. (A and B) IF results for RIPK3 and MLKL in thymus tissues ( n = 20 broilers per group). (C and D) The detection for mRNA and protein levels of necroptosis factors (RIPK1, RIPK3, Caspase8, MLKL, and P -MLKL) in thymus tissues ( n = 20 broilers per group). (E and F) IF results for necroptosis marker factors (RIPK3 and MLKL) in MSB-1 cells and quantitative analysis ( n = 3 independent cell culture experiments). (G and I) The transcription and protein levels of necroptosis factors, including RIPK1, RIPK3, Caspase8, MLKL, and P -MLKL, in MSB-1 cells ( n = 3 independent cell culture experiments). All the data are displayed as the means ± SD ( n = 3). The statistically significant differences are not denoted with the same letters, such as ‘a, b, c, and d’.

    Journal: Redox Report : Communications in Free Radical Research

    Article Title: Elucidating the protective role of quercetin against lipopolysaccharide-induced necroptosis in broiler thymus: insights from Nrf2/PERK signaling based on network pharmacology and experimental validation

    doi: 10.1080/13510002.2026.2663621

    Figure Lengend Snippet: QUE attenuated necroptosis through Nrf2/PERK under LPS exposure. (A and B) IF results for RIPK3 and MLKL in thymus tissues ( n = 20 broilers per group). (C and D) The detection for mRNA and protein levels of necroptosis factors (RIPK1, RIPK3, Caspase8, MLKL, and P -MLKL) in thymus tissues ( n = 20 broilers per group). (E and F) IF results for necroptosis marker factors (RIPK3 and MLKL) in MSB-1 cells and quantitative analysis ( n = 3 independent cell culture experiments). (G and I) The transcription and protein levels of necroptosis factors, including RIPK1, RIPK3, Caspase8, MLKL, and P -MLKL, in MSB-1 cells ( n = 3 independent cell culture experiments). All the data are displayed as the means ± SD ( n = 3). The statistically significant differences are not denoted with the same letters, such as ‘a, b, c, and d’.

    Article Snippet: Following dewaxing, the slices were subjected to antigen retrieval and blocked with 3% BSA at room temperature for 30 min. Then, the slices were incubated with PERK (GB11507-100, 1:200, Rabbit pAb, Servicebio, Wuhan), MLKL (GB155699-100, 1:5000, Rabbit pAb, Servicebio, Wuhan), and RIPK3 (bs-3551R, 1:100, Rabbit pAb, Bioss, Beijing) primary antibodies, secondary antibodies, and stained with DAPI solution.

    Techniques: Marker, Cell Culture

    TBK1 and NF-κB positively regulate IRG1 levels in HMPV-infected human macrophages. MDMs were incubated with 5 or 10 μM of the TBK1 inhibitor BX795 for 30 min before infection with HMPV for 24 h. ( A ) IRG1 and IFN-β mRNA levels ( n ≥ 3) were analysed by qRT-PCR (left panels), while in panel ( B ) IRG1 and GAPDH protein levels were determined by immunoblotting, quantified, and presented with SD relative to uninfected cells treated with DMSO ( n = 2; right panel). (C–K) MDMs were transfected with siRNAs targeting the NF-κB subunit RELA (C–E), IRF1 (F–H), RIPK3 (I–K), or control siRNA (siNTC) before infection with HMPV for 6, 9, or 24 h and analysis of IRG1 mRNA levels by qRT-PCR or IRG1, RelA/p65, IRF1, RIPK3, or GAPDH protein levels by immunoblotting. IRG1 mRNA levels were assessed relative to siNTC-transfected, uninfected MDMs for all experiments. ( C, E ) IRG1 mRNA after 6 h ( n = 3) or 24 h HMPV ( n = 3). ( D ) Protein levels of IRG1, RelA/p65, and GAPDH ( n = 3) after 9 h HMPV. ( F, H ) IRG1 mRNA after 6 h ( n = 4) or 24 h of infection ( n = 3). ( G ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( I, K ) IRG1 mRNA after 6 h ( n = 3) or 24 h of infection ( n = 3). ( J ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( L ) MDMs were incubated with 100 nM of the RIPK3 inhibitor GSK 872 for 1 h before infection with HMPV for 24 h. IRG1 mRNA levels were normalized relative to uninfected cells treated with DMSO ( n = 2). Single comparison between control (NTC) siRNA and target-siRNA conditions were calculated using paired t -test with Tukey post-hoc test. * P < .05, ** P < .01, *** P < .001, **** P < .0001; ns = non-significant. See also

    Journal: NAR Molecular Medicine

    Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

    doi: 10.1093/narmme/ugag017

    Figure Lengend Snippet: TBK1 and NF-κB positively regulate IRG1 levels in HMPV-infected human macrophages. MDMs were incubated with 5 or 10 μM of the TBK1 inhibitor BX795 for 30 min before infection with HMPV for 24 h. ( A ) IRG1 and IFN-β mRNA levels ( n ≥ 3) were analysed by qRT-PCR (left panels), while in panel ( B ) IRG1 and GAPDH protein levels were determined by immunoblotting, quantified, and presented with SD relative to uninfected cells treated with DMSO ( n = 2; right panel). (C–K) MDMs were transfected with siRNAs targeting the NF-κB subunit RELA (C–E), IRF1 (F–H), RIPK3 (I–K), or control siRNA (siNTC) before infection with HMPV for 6, 9, or 24 h and analysis of IRG1 mRNA levels by qRT-PCR or IRG1, RelA/p65, IRF1, RIPK3, or GAPDH protein levels by immunoblotting. IRG1 mRNA levels were assessed relative to siNTC-transfected, uninfected MDMs for all experiments. ( C, E ) IRG1 mRNA after 6 h ( n = 3) or 24 h HMPV ( n = 3). ( D ) Protein levels of IRG1, RelA/p65, and GAPDH ( n = 3) after 9 h HMPV. ( F, H ) IRG1 mRNA after 6 h ( n = 4) or 24 h of infection ( n = 3). ( G ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( I, K ) IRG1 mRNA after 6 h ( n = 3) or 24 h of infection ( n = 3). ( J ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( L ) MDMs were incubated with 100 nM of the RIPK3 inhibitor GSK 872 for 1 h before infection with HMPV for 24 h. IRG1 mRNA levels were normalized relative to uninfected cells treated with DMSO ( n = 2). Single comparison between control (NTC) siRNA and target-siRNA conditions were calculated using paired t -test with Tukey post-hoc test. * P < .05, ** P < .01, *** P < .001, **** P < .0001; ns = non-significant. See also

    Article Snippet: The primary human antibodies IRG1, IRF1, RIPK3, NF-κB p65, phospho-STAT1 (Tyr701), STAT1, and Nrf2 were purchased from Cell Signalling Technology.

    Techniques: Infection, Incubation, Quantitative RT-PCR, Western Blot, Transfection, Control, Comparison