anti rnf20  (Cell Signaling Technology Inc)


Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Cell Signaling Technology Inc anti rnf20
    Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rnf20/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rnf20 - by Bioz Stars, 2024-06
    86/100 stars

    Images

    anti rnf20  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Cell Signaling Technology Inc anti rnf20
    Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rnf20/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rnf20 - by Bioz Stars, 2024-06
    86/100 stars

    Images

    anti rnf20  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Cell Signaling Technology Inc anti rnf20
    ( A ) NCOA4 interacts with <t>RNF20</t> as determined by Coomassie bright blue staining. IgG, immunoglobulin G; IP, immunoprecipitation; WCL, whole-cell lysate. ( B ) Interaction between endogenous NCOA4 and RNF20 in C2C12 cells was confirmed by Co-IP with Western blotting ( n = 9). ( C ) Western blot analysis of RNF20 and H2Bub1 expression in C2C12 cells treated with 0, 10, 15, 20, 25, and 30 μM DFO in differentiation medium for 3 days ( n = 9). ( D ) Western blot analysis of RNF20 and H2Bub1 in C2C12 cells treated as indicated in differentiation medium for 3 days ( n = 9). ( E ) Western blot analysis of NCOA4, RNF20, and H2Bub1 in C2C12 cells treated as indicated in differentiation medium for 3 days ( n = 9). The results represent the means ± SD. * P < 0.05 and ** P < 0.01.
    Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rnf20/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rnf20 - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Iron deficiency–induced ferritinophagy impairs skeletal muscle regeneration through RNF20-mediated H2Bub1 modification"

    Article Title: Iron deficiency–induced ferritinophagy impairs skeletal muscle regeneration through RNF20-mediated H2Bub1 modification

    Journal: Science Advances

    doi: 10.1126/sciadv.adf4345

    ( A ) NCOA4 interacts with RNF20 as determined by Coomassie bright blue staining. IgG, immunoglobulin G; IP, immunoprecipitation; WCL, whole-cell lysate. ( B ) Interaction between endogenous NCOA4 and RNF20 in C2C12 cells was confirmed by Co-IP with Western blotting ( n = 9). ( C ) Western blot analysis of RNF20 and H2Bub1 expression in C2C12 cells treated with 0, 10, 15, 20, 25, and 30 μM DFO in differentiation medium for 3 days ( n = 9). ( D ) Western blot analysis of RNF20 and H2Bub1 in C2C12 cells treated as indicated in differentiation medium for 3 days ( n = 9). ( E ) Western blot analysis of NCOA4, RNF20, and H2Bub1 in C2C12 cells treated as indicated in differentiation medium for 3 days ( n = 9). The results represent the means ± SD. * P < 0.05 and ** P < 0.01.
    Figure Legend Snippet: ( A ) NCOA4 interacts with RNF20 as determined by Coomassie bright blue staining. IgG, immunoglobulin G; IP, immunoprecipitation; WCL, whole-cell lysate. ( B ) Interaction between endogenous NCOA4 and RNF20 in C2C12 cells was confirmed by Co-IP with Western blotting ( n = 9). ( C ) Western blot analysis of RNF20 and H2Bub1 expression in C2C12 cells treated with 0, 10, 15, 20, 25, and 30 μM DFO in differentiation medium for 3 days ( n = 9). ( D ) Western blot analysis of RNF20 and H2Bub1 in C2C12 cells treated as indicated in differentiation medium for 3 days ( n = 9). ( E ) Western blot analysis of NCOA4, RNF20, and H2Bub1 in C2C12 cells treated as indicated in differentiation medium for 3 days ( n = 9). The results represent the means ± SD. * P < 0.05 and ** P < 0.01.

    Techniques Used: Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Expressing

    ( A ) mRNA expression of RNF20 , MyHC , MyoD , and MyoG was measured by qRT-PCR after knockdown of RNF20 by siRNA in C2C12 cells cultured for 3 days in differentiation medium ( n = 9). ( B ) Western blot analysis of RNF20, H2Bub1, MyHC, MyoD, and MyoG protein expression in the RNF20 knockdown or control group of C2C12 cells cultured for 3 days in differentiation medium ( n = 9). ( C ) Immunofluorescence staining for MyHC in the RNF20 knockdown and control group of C2C12 cells cultured for 3 days in differentiation medium ( n = 9). Scale bars, 100 μm. ( D ) mRNA expression of RNF20 , MyHC , MyoD , and MyoG in the RNF20 overexpression group of C2C12 cells treated with or without DFO (20 μM) ( n = 9). ( E ) Western blot analysis of RNF20, H2Bub1, MyHC, MyoD and MyoG expression in the RNF20 overexpression group of C2C12 cells treated with or without DFO (20 μM) ( n = 9). ( F ) Immunofluorescence staining for MyHC in the RNF20 overexpression group of C2C12 cells treated with or without DFO (20 μM) ( n = 9). Scale bars, 100 μm. ( G ) Results of ChIP-qPCR confirmed changes in H2Bub1 modification and binding to the promoter of MyoD and MyoG in control and iron-deficient C2C12 cells treated with or without DC661 following the induction of differentiation for 3 days ( n = 9). ( H ) ChIP-qPCR analysis of changes in H2Bub1 binding to the promoters of MyoD and MyoG in RNF20-overexpressing C2C12 cells under DFO (20 μM), treated or untreated ( n = 9). The results represent the means ± SD. * P < 0.05 and ** P < 0.01. ID, iron-deficient.
    Figure Legend Snippet: ( A ) mRNA expression of RNF20 , MyHC , MyoD , and MyoG was measured by qRT-PCR after knockdown of RNF20 by siRNA in C2C12 cells cultured for 3 days in differentiation medium ( n = 9). ( B ) Western blot analysis of RNF20, H2Bub1, MyHC, MyoD, and MyoG protein expression in the RNF20 knockdown or control group of C2C12 cells cultured for 3 days in differentiation medium ( n = 9). ( C ) Immunofluorescence staining for MyHC in the RNF20 knockdown and control group of C2C12 cells cultured for 3 days in differentiation medium ( n = 9). Scale bars, 100 μm. ( D ) mRNA expression of RNF20 , MyHC , MyoD , and MyoG in the RNF20 overexpression group of C2C12 cells treated with or without DFO (20 μM) ( n = 9). ( E ) Western blot analysis of RNF20, H2Bub1, MyHC, MyoD and MyoG expression in the RNF20 overexpression group of C2C12 cells treated with or without DFO (20 μM) ( n = 9). ( F ) Immunofluorescence staining for MyHC in the RNF20 overexpression group of C2C12 cells treated with or without DFO (20 μM) ( n = 9). Scale bars, 100 μm. ( G ) Results of ChIP-qPCR confirmed changes in H2Bub1 modification and binding to the promoter of MyoD and MyoG in control and iron-deficient C2C12 cells treated with or without DC661 following the induction of differentiation for 3 days ( n = 9). ( H ) ChIP-qPCR analysis of changes in H2Bub1 binding to the promoters of MyoD and MyoG in RNF20-overexpressing C2C12 cells under DFO (20 μM), treated or untreated ( n = 9). The results represent the means ± SD. * P < 0.05 and ** P < 0.01. ID, iron-deficient.

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Immunofluorescence, Staining, Over Expression, Modification, Binding Assay

    Mice were divided into the following four groups with nine animals per group: normal control group (NC), iron-deficient group, ID plus 3-MA (ID + 3-MA) group, and ID plus RNF20 overexpression (ID + OE-RNF20) group. ( A ) Iron levels in the skeletal muscle tissue were measured via LA-ICP-MS and in situ imaging analysis. ( B ) Representative images of GA muscles in NC mice, iron-deficient mice, ID + 3-MA mice, and ID + OE-RNF20 mice. ( C ) Skeletal function was estimated by tests of grip strength, hanging time, and exhaustion time ( n = 9). ( D ) H&E staining and Prussian blue staining for iron in the GA muscle of different groups of mice ( n = 9). Scale bars in H&E staining, 100 μm. Scale bars in Prussian blue staining, 50 μm. ( E ) Western blot analysis of MyoD and MyoG expression in GA muscle samples from different groups of mice ( n = 9). ( F to H ) Immunofluorescence analysis of MyoD and MyoG expression in the GA muscle of different groups of mice ( n = 9). Scale bar, 50 μm. The results represent the means ± SD. * P < 0.05 and ** P < 0.01. ID, iron-deficient.
    Figure Legend Snippet: Mice were divided into the following four groups with nine animals per group: normal control group (NC), iron-deficient group, ID plus 3-MA (ID + 3-MA) group, and ID plus RNF20 overexpression (ID + OE-RNF20) group. ( A ) Iron levels in the skeletal muscle tissue were measured via LA-ICP-MS and in situ imaging analysis. ( B ) Representative images of GA muscles in NC mice, iron-deficient mice, ID + 3-MA mice, and ID + OE-RNF20 mice. ( C ) Skeletal function was estimated by tests of grip strength, hanging time, and exhaustion time ( n = 9). ( D ) H&E staining and Prussian blue staining for iron in the GA muscle of different groups of mice ( n = 9). Scale bars in H&E staining, 100 μm. Scale bars in Prussian blue staining, 50 μm. ( E ) Western blot analysis of MyoD and MyoG expression in GA muscle samples from different groups of mice ( n = 9). ( F to H ) Immunofluorescence analysis of MyoD and MyoG expression in the GA muscle of different groups of mice ( n = 9). Scale bar, 50 μm. The results represent the means ± SD. * P < 0.05 and ** P < 0.01. ID, iron-deficient.

    Techniques Used: Over Expression, In Situ, Imaging, Muscles, Staining, Western Blot, Expressing, Immunofluorescence

    ( A and B ) Immunofluorescence staining for RNF20 and H2Bub1 in different groups of mice. The mean fluorescence intensities of RNF20 and H2Bub1 are shown below ( n = 9). Scale bars, 50 μm. ( C ) Western blot analysis of RNF20 and H2Bub1 expression in different groups of mice ( n = 9). The results represent the means ± SD. ** P < 0.01. ID, iron-deficient.
    Figure Legend Snippet: ( A and B ) Immunofluorescence staining for RNF20 and H2Bub1 in different groups of mice. The mean fluorescence intensities of RNF20 and H2Bub1 are shown below ( n = 9). Scale bars, 50 μm. ( C ) Western blot analysis of RNF20 and H2Bub1 expression in different groups of mice ( n = 9). The results represent the means ± SD. ** P < 0.01. ID, iron-deficient.

    Techniques Used: Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing

    The mice were divided into the following four groups with nine mice per group: NC, iron-deficient group, ID + 3-MA group, and ID + OE-RNF20 group. The CTX was injected into GA muscles of the above mice to induce acute injury. The injected GA muscles were harvested at the 7 day post-CTX injury (dpi) for the assessment of the regeneration process. ( A ) H&E staining of the GA muscle and the average myofiber CSA in different groups of mice at 7 dpi ( n = 9). Scale bars, 100 μm. ( B ) Western blot analysis of eMyHC, MyoD, and MyoG expression in different groups of mice at 7 dpi ( n = 9). ( C ) Immunofluorescence analysis of eMyHC + fibers in the GA muscle of different groups of mice at 7 dpi. Scale bars, 50 μm. ( D ) Percent distribution of the muscle fiber cross-sectional area derived from the different groups of mice at 7 dpi ( n = 9). ( E ) eMyHC + cells and peripherally nucleated fibers in the GA muscle of different groups of mice at 7 dpi ( n = 9). ( F to H ) Immunofluorescence analysis of MyoD + and MyoG + cells in the GA muscle of different groups of mice at 7 dpi ( n = 9). Scale bars, 20 μm. The results represent the means ± SD. * P < 0.05 and ** P < 0.01. ID, iron-deficient.
    Figure Legend Snippet: The mice were divided into the following four groups with nine mice per group: NC, iron-deficient group, ID + 3-MA group, and ID + OE-RNF20 group. The CTX was injected into GA muscles of the above mice to induce acute injury. The injected GA muscles were harvested at the 7 day post-CTX injury (dpi) for the assessment of the regeneration process. ( A ) H&E staining of the GA muscle and the average myofiber CSA in different groups of mice at 7 dpi ( n = 9). Scale bars, 100 μm. ( B ) Western blot analysis of eMyHC, MyoD, and MyoG expression in different groups of mice at 7 dpi ( n = 9). ( C ) Immunofluorescence analysis of eMyHC + fibers in the GA muscle of different groups of mice at 7 dpi. Scale bars, 50 μm. ( D ) Percent distribution of the muscle fiber cross-sectional area derived from the different groups of mice at 7 dpi ( n = 9). ( E ) eMyHC + cells and peripherally nucleated fibers in the GA muscle of different groups of mice at 7 dpi ( n = 9). ( F to H ) Immunofluorescence analysis of MyoD + and MyoG + cells in the GA muscle of different groups of mice at 7 dpi ( n = 9). Scale bars, 20 μm. The results represent the means ± SD. * P < 0.05 and ** P < 0.01. ID, iron-deficient.

    Techniques Used: Injection, Muscles, Staining, Western Blot, Expressing, Immunofluorescence, Derivative Assay

    The mice were divided into the following four groups with nine mice per group: NC group (NCOA4 fl/fl ), cKO group (Pax7 CreER , NCOA4 fl/fl ), iron-deficient group (NCOA4 fl/fl ) group, and ID + cKO group (Pax7 CreER , NCOA4 fl/fl ) group. ( A ) Schematic diagram of the procedures for iron deficiency induction, NCOA4 cKO, and CTX-induced muscle injury. ( B ) Schematic diagram of SC isolation. ( C ) Western blot analysis of NCOA4, RNF20, and H2Bub1 protein levels in GA muscle samples from different groups of mice at 7 dpi ( n = 9). ( D ) Representative images of myofibers isolated from the extensor digitorum longus (EDL) muscle of NCOA4 SCs/WT and NCOA4 SCs/KO mice ( n = 9). Immunofluorescence staining of Pax7 (pink), NCOA4 (red), RNF20 (green), and DAPI (blue). Scale bars, 20 μm. ( E ) Representative images of myofibers isolated from the EDL muscle of NCOA4 SCs/WT and NCOA4 SCs/KO mice ( n = 9). Immunofluorescence of Pax7 (pink), NCOA4 (red), H2Bub1 (green), and DAPI (blue) staining. Scale bars, 20 μm. The results represent the means ± SD. * P < 0.05 and ** P < 0.01. ID, iron-deficient.
    Figure Legend Snippet: The mice were divided into the following four groups with nine mice per group: NC group (NCOA4 fl/fl ), cKO group (Pax7 CreER , NCOA4 fl/fl ), iron-deficient group (NCOA4 fl/fl ) group, and ID + cKO group (Pax7 CreER , NCOA4 fl/fl ) group. ( A ) Schematic diagram of the procedures for iron deficiency induction, NCOA4 cKO, and CTX-induced muscle injury. ( B ) Schematic diagram of SC isolation. ( C ) Western blot analysis of NCOA4, RNF20, and H2Bub1 protein levels in GA muscle samples from different groups of mice at 7 dpi ( n = 9). ( D ) Representative images of myofibers isolated from the extensor digitorum longus (EDL) muscle of NCOA4 SCs/WT and NCOA4 SCs/KO mice ( n = 9). Immunofluorescence staining of Pax7 (pink), NCOA4 (red), RNF20 (green), and DAPI (blue). Scale bars, 20 μm. ( E ) Representative images of myofibers isolated from the EDL muscle of NCOA4 SCs/WT and NCOA4 SCs/KO mice ( n = 9). Immunofluorescence of Pax7 (pink), NCOA4 (red), H2Bub1 (green), and DAPI (blue) staining. Scale bars, 20 μm. The results represent the means ± SD. * P < 0.05 and ** P < 0.01. ID, iron-deficient.

    Techniques Used: Isolation, Western Blot, Immunofluorescence, Staining

    anti rnf20  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Cell Signaling Technology Inc anti rnf20
    KSHV RTA interacts with the host E3 ubiquitin ligase complex <t>RNF20/40.</t> (A) FLAG immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (B) RNF20 or RNF40 immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (C) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA (3xF-RTA), and the samples were subjected to immunoblot analysis. (D) 3xFLAG-tagged RTA, RNF20, and RNF40 were translated with TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI). The expressed proteins were then subjected to immunoprecipitation and immunoblot analysis. (E) In situ PLA using proximity probes against RTA and RNF20 was performed to visualize their interaction in iBCBL1-3xFLAG-RTA cells reactivated for 12 h. (F) Model representation of the full-length (FL) KSHV RTA and its truncation mutants. RING, RING-like domain; LR, leucine heptapeptide repeat; TAD, transactivation domain; and NLS, nuclear localization signal. (G) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA full-length (FL) and truncation mutants, and the samples were subjected to immunoblot analysis.
    Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rnf20/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rnf20 - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation"

    Article Title: KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation

    Journal: Journal of Virology

    doi: 10.1128/jvi.01389-23

    KSHV RTA interacts with the host E3 ubiquitin ligase complex RNF20/40. (A) FLAG immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (B) RNF20 or RNF40 immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (C) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA (3xF-RTA), and the samples were subjected to immunoblot analysis. (D) 3xFLAG-tagged RTA, RNF20, and RNF40 were translated with TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI). The expressed proteins were then subjected to immunoprecipitation and immunoblot analysis. (E) In situ PLA using proximity probes against RTA and RNF20 was performed to visualize their interaction in iBCBL1-3xFLAG-RTA cells reactivated for 12 h. (F) Model representation of the full-length (FL) KSHV RTA and its truncation mutants. RING, RING-like domain; LR, leucine heptapeptide repeat; TAD, transactivation domain; and NLS, nuclear localization signal. (G) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA full-length (FL) and truncation mutants, and the samples were subjected to immunoblot analysis.
    Figure Legend Snippet: KSHV RTA interacts with the host E3 ubiquitin ligase complex RNF20/40. (A) FLAG immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (B) RNF20 or RNF40 immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (C) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA (3xF-RTA), and the samples were subjected to immunoblot analysis. (D) 3xFLAG-tagged RTA, RNF20, and RNF40 were translated with TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI). The expressed proteins were then subjected to immunoprecipitation and immunoblot analysis. (E) In situ PLA using proximity probes against RTA and RNF20 was performed to visualize their interaction in iBCBL1-3xFLAG-RTA cells reactivated for 12 h. (F) Model representation of the full-length (FL) KSHV RTA and its truncation mutants. RING, RING-like domain; LR, leucine heptapeptide repeat; TAD, transactivation domain; and NLS, nuclear localization signal. (G) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA full-length (FL) and truncation mutants, and the samples were subjected to immunoblot analysis.

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, In Situ

    RNF20 promotes KSHV lytic cycle in iBCBL1-3xFLAG-RTA cell line. (A) Immunoblot analysis of RNF20/40 and viral proteins in cells transduced with lenti-shControl or lenti-shRNF20 for 2 days and then reactivated for various time points. E: early viral protein and L: late viral protein. (B) MTT assay was performed to measure cell viability of cells transduced with lenti-shControl or lenti-shRNF20 at 3 dpi. (C) Viral gene expression measured by RT-qPCR at various time points. The fold change is calculated relative to shControl 0 hpi. IE: immediate early genes; E: early genes; and L: late genes. (D) Viral DNA level at various time points measured by qPCR. The fold change is calculated relative to shControl 0 hpi. (E) KSHV production was determined by transferring the virus-containing supernatant from reactivated lenti-shRNA transduced iBCBL1-3xFLAG-RTA to 293T cells. Total DNA in infected 293T cells was analyzed at 2 hpi. The amount of viral DNA was quantified by qPCR and normalized to host DNA. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 and n.s.: not significant (sample of n = 3).
    Figure Legend Snippet: RNF20 promotes KSHV lytic cycle in iBCBL1-3xFLAG-RTA cell line. (A) Immunoblot analysis of RNF20/40 and viral proteins in cells transduced with lenti-shControl or lenti-shRNF20 for 2 days and then reactivated for various time points. E: early viral protein and L: late viral protein. (B) MTT assay was performed to measure cell viability of cells transduced with lenti-shControl or lenti-shRNF20 at 3 dpi. (C) Viral gene expression measured by RT-qPCR at various time points. The fold change is calculated relative to shControl 0 hpi. IE: immediate early genes; E: early genes; and L: late genes. (D) Viral DNA level at various time points measured by qPCR. The fold change is calculated relative to shControl 0 hpi. (E) KSHV production was determined by transferring the virus-containing supernatant from reactivated lenti-shRNA transduced iBCBL1-3xFLAG-RTA to 293T cells. Total DNA in infected 293T cells was analyzed at 2 hpi. The amount of viral DNA was quantified by qPCR and normalized to host DNA. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 and n.s.: not significant (sample of n = 3).

    Techniques Used: Western Blot, Transduction, MTT Assay, Expressing, Quantitative RT-PCR, Transferring, Virus, shRNA, Infection

    RNF20 is necessary for inducing RTA expression during lytic KSHV reactivation. (A) Immunoblot analysis of RNF20/40 and viral proteins in BCBL1 cells transduced with lenti-shControl or lenti-shRNF20 for 2 days and then reactivated with TPA for various time points. IE: immediate early protein; E: early protein; and L: late protein. (B) Viral gene expression calculated relative to shControl 0 hpi was measured by RT-qPCR at various time points. IE: immediate early genes; E: early genes; and L: late genes. The degree of statistical significance in the reduction of lytic gene expression by shRNF20 was calculated by t-tests (*P ≤ 0.05 and n = 3).
    Figure Legend Snippet: RNF20 is necessary for inducing RTA expression during lytic KSHV reactivation. (A) Immunoblot analysis of RNF20/40 and viral proteins in BCBL1 cells transduced with lenti-shControl or lenti-shRNF20 for 2 days and then reactivated with TPA for various time points. IE: immediate early protein; E: early protein; and L: late protein. (B) Viral gene expression calculated relative to shControl 0 hpi was measured by RT-qPCR at various time points. IE: immediate early genes; E: early genes; and L: late genes. The degree of statistical significance in the reduction of lytic gene expression by shRNF20 was calculated by t-tests (*P ≤ 0.05 and n = 3).

    Techniques Used: Expressing, Western Blot, Transduction, Quantitative RT-PCR

    KSHV RTA utilizes RNF20/40 to induce cellular gene expression. (A) Host gene expression measured by RT-qPCR at 12 hpi using the iBCBL1-3xFLAG-RTA samples shown in Fig. 3. (B) Immunoblot analysis of RNF20 and KSHV RTA in iSLK cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then Dox was added to induce RTA expression. (C) MTT assay was performed to measure cell viability of cells transduced with lenti-shControl or lenti-shRNF20 at 3 dpi. (D) Host gene expression measured by RT-qPCR at 12 hpi. The degree of statistical significance in the reduction of host gene expression by shRNF20 was calculated by t-tests (*P ≤ 0.05, n.s.: not significant, and n = 3).
    Figure Legend Snippet: KSHV RTA utilizes RNF20/40 to induce cellular gene expression. (A) Host gene expression measured by RT-qPCR at 12 hpi using the iBCBL1-3xFLAG-RTA samples shown in Fig. 3. (B) Immunoblot analysis of RNF20 and KSHV RTA in iSLK cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then Dox was added to induce RTA expression. (C) MTT assay was performed to measure cell viability of cells transduced with lenti-shControl or lenti-shRNF20 at 3 dpi. (D) Host gene expression measured by RT-qPCR at 12 hpi. The degree of statistical significance in the reduction of host gene expression by shRNF20 was calculated by t-tests (*P ≤ 0.05, n.s.: not significant, and n = 3).

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transduction, MTT Assay

    KSHV RTA uses RNF20/40 to induce KSHV lytic promoters. (A) 293T cells were transfected with a KSHV ORF50 promoter luciferase reporter plasmid along with RTA, RNF20, and RNF40. Luciferase activity was measured, and the results were normalized to vector control. (B) 293T cells were transfected with a CMV promoter luciferase reporter plasmid along with RTA, RNF20, and RNF40. Luciferase activity was measured, and the results were normalized to vector control. (C) 293T cells were transfected with a luciferase reporter plasmid containing a KSHV promoter (ORF50, ORF57, or K2) along with RNF20/40 and an RTA WT or mutant. Luciferase activity was measured and normalized to vector control. (D) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV ORF50 promoter luciferase reporter plasmid along with KSHV RTA. (E) Luciferase activity of samples described in panel (D). Results were normalized to shControl vector. (F) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV ORF57 promoter luciferase reporter plasmid along with KSHV RTA. (G) Luciferase activity of samples described in panel (F). Results normalized to shControl vector. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).
    Figure Legend Snippet: KSHV RTA uses RNF20/40 to induce KSHV lytic promoters. (A) 293T cells were transfected with a KSHV ORF50 promoter luciferase reporter plasmid along with RTA, RNF20, and RNF40. Luciferase activity was measured, and the results were normalized to vector control. (B) 293T cells were transfected with a CMV promoter luciferase reporter plasmid along with RTA, RNF20, and RNF40. Luciferase activity was measured, and the results were normalized to vector control. (C) 293T cells were transfected with a luciferase reporter plasmid containing a KSHV promoter (ORF50, ORF57, or K2) along with RNF20/40 and an RTA WT or mutant. Luciferase activity was measured and normalized to vector control. (D) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV ORF50 promoter luciferase reporter plasmid along with KSHV RTA. (E) Luciferase activity of samples described in panel (D). Results were normalized to shControl vector. (F) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV ORF57 promoter luciferase reporter plasmid along with KSHV RTA. (G) Luciferase activity of samples described in panel (F). Results normalized to shControl vector. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).

    Techniques Used: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Mutagenesis, Western Blot, Transduction

    RTA binding to its target promoters is necessary for the synergistic promoter induction by RTA and RNF20/40. (A) 293T cells were transfected with a luciferase reporter plasmid containing different regions of the KSHV ORF50 promoter along with RTA with or without RNF20/40. Luciferase activity was measured, and the results were normalized to vector control. (B) Schematic of promoter luciferase reporters. WT and a mutant RTA response element (RRE) derived from ORF57 promoter were cloned into the luciferase reporter plasmid pGL4.27 containing a minimal promoter, which has only a TATA-box promoter element. (C) Luciferase activity of 293T cells co-transfected with RRE WT or mutant luciferase reporter plasmid with WT or mutant RTAs. Results were normalized to vector-transfected cells. (D) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV RRE luciferase reporter plasmid along with KSHV RTA. (E) Luciferase activity of samples described in panel (D). Results were normalized to shControl sample. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).
    Figure Legend Snippet: RTA binding to its target promoters is necessary for the synergistic promoter induction by RTA and RNF20/40. (A) 293T cells were transfected with a luciferase reporter plasmid containing different regions of the KSHV ORF50 promoter along with RTA with or without RNF20/40. Luciferase activity was measured, and the results were normalized to vector control. (B) Schematic of promoter luciferase reporters. WT and a mutant RTA response element (RRE) derived from ORF57 promoter were cloned into the luciferase reporter plasmid pGL4.27 containing a minimal promoter, which has only a TATA-box promoter element. (C) Luciferase activity of 293T cells co-transfected with RRE WT or mutant luciferase reporter plasmid with WT or mutant RTAs. Results were normalized to vector-transfected cells. (D) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV RRE luciferase reporter plasmid along with KSHV RTA. (E) Luciferase activity of samples described in panel (D). Results were normalized to shControl sample. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).

    Techniques Used: Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Mutagenesis, Derivative Assay, Clone Assay, Western Blot, Transduction

    Overexpression of RNF20/40 further promotes RTA-induced lytic viral gene expression. (A) Immunoblot analysis of 293TBAC16 cells co-transfected with KSHV RTA and RNF20/40 WT or RING domain deletion mutant. (B) Viral gene expression was measured by RT-qPCR. The degree of statistical significance is calculated by t-tests (*P ≤ 0.05 and n = 3).
    Figure Legend Snippet: Overexpression of RNF20/40 further promotes RTA-induced lytic viral gene expression. (A) Immunoblot analysis of 293TBAC16 cells co-transfected with KSHV RTA and RNF20/40 WT or RING domain deletion mutant. (B) Viral gene expression was measured by RT-qPCR. The degree of statistical significance is calculated by t-tests (*P ≤ 0.05 and n = 3).

    Techniques Used: Over Expression, Expressing, Western Blot, Transfection, Mutagenesis, Quantitative RT-PCR

    RNF20 is recruited to RTA binding sites on viral and host genomes during lytic reactivation of KSHV. (A) Co-localization of RNF20 with LANA-marked KSHV episomes was determined in iSLKBAC16-3xFLAG-LANA cells using immunofluorescence analysis of RNF20 and FLAG (LANA) during latency (0 hpi) and lytic reactivation (24 hpi). DAPI: nucleus; green: RNF20; and red: LANA. (B) Representative LANA puncta were connected by yellow line, and the co-localization of LANA with KDM2B along the line was analyzed by ImageJ. (C) CUT&RUN assay was performed on iBCBL1-3xFLAG RTA cells reactivated for 12 h to examine RTA and RNF20 binding to viral and host gene promoters.
    Figure Legend Snippet: RNF20 is recruited to RTA binding sites on viral and host genomes during lytic reactivation of KSHV. (A) Co-localization of RNF20 with LANA-marked KSHV episomes was determined in iSLKBAC16-3xFLAG-LANA cells using immunofluorescence analysis of RNF20 and FLAG (LANA) during latency (0 hpi) and lytic reactivation (24 hpi). DAPI: nucleus; green: RNF20; and red: LANA. (B) Representative LANA puncta were connected by yellow line, and the co-localization of LANA with KDM2B along the line was analyzed by ImageJ. (C) CUT&RUN assay was performed on iBCBL1-3xFLAG RTA cells reactivated for 12 h to examine RTA and RNF20 binding to viral and host gene promoters.

    Techniques Used: Binding Assay, Immunofluorescence

    RNF20 regulates RNA polymerase II occupancy on RTA promoter during lytic reactivation of KSHV. (A–D) iBCBL1-3xFLAG-RTA cells were transduced with lenti-shControl or lenti-shRNF20 then reactivated for 12 h, and ChIP-qPCR analysis was performed at the indicated sites of the RTA promoter and gene body. (A) H2BK120ub ChIP. (B) H3K4me3 ChIP. (C) RNA polymerase II (RNAP II) ChIP. (D) ChIP for RNAP II Serine 2 phosphorylation. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).
    Figure Legend Snippet: RNF20 regulates RNA polymerase II occupancy on RTA promoter during lytic reactivation of KSHV. (A–D) iBCBL1-3xFLAG-RTA cells were transduced with lenti-shControl or lenti-shRNF20 then reactivated for 12 h, and ChIP-qPCR analysis was performed at the indicated sites of the RTA promoter and gene body. (A) H2BK120ub ChIP. (B) H3K4me3 ChIP. (C) RNA polymerase II (RNAP II) ChIP. (D) ChIP for RNAP II Serine 2 phosphorylation. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).

    Techniques Used: Transduction

    Working model for RTA re-tasking RNF20/40 to promote the KSHV lytic cycle. Upon KSHV lytic reactivation, once RTA is expressed, RTA binds to its own gene promoter via interacting with RBPJk and recruits RNF20/40 to help autoregulate and sustain RTA expression. Our results suggest that RNF20/40 facilitates the recruitment of transcriptionally active RNAP II onto RTA gene resulting in increased viral gene expression. RTA binds to its viral and host target promoters, directly or indirectly, and uses RNF20/40 to promote the expression of its viral and host target genes as well. Created with BioRender.com.
    Figure Legend Snippet: Working model for RTA re-tasking RNF20/40 to promote the KSHV lytic cycle. Upon KSHV lytic reactivation, once RTA is expressed, RTA binds to its own gene promoter via interacting with RBPJk and recruits RNF20/40 to help autoregulate and sustain RTA expression. Our results suggest that RNF20/40 facilitates the recruitment of transcriptionally active RNAP II onto RTA gene resulting in increased viral gene expression. RTA binds to its viral and host target promoters, directly or indirectly, and uses RNF20/40 to promote the expression of its viral and host target genes as well. Created with BioRender.com.

    Techniques Used: Expressing

    anti rnf20  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Cell Signaling Technology Inc anti rnf20
    KSHV RTA interacts with the host E3 ubiquitin ligase complex <t>RNF20/40.</t> (A) FLAG immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (B) RNF20 or RNF40 immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (C) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA (3xF-RTA), and the samples were subjected to immunoblot analysis. (D) 3xFLAG-tagged RTA, RNF20, and RNF40 were translated with TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI). The expressed proteins were then subjected to immunoprecipitation and immunoblot analysis. (E) In situ PLA using proximity probes against RTA and RNF20 was performed to visualize their interaction in iBCBL1-3xFLAG-RTA cells reactivated for 12 h. (F) Model representation of the full-length (FL) KSHV RTA and its truncation mutants. RING, RING-like domain; LR, leucine heptapeptide repeat; TAD, transactivation domain; and NLS, nuclear localization signal. (G) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA full-length (FL) and truncation mutants, and the samples were subjected to immunoblot analysis.
    Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rnf20/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rnf20 - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation"

    Article Title: KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation

    Journal: Journal of Virology

    doi: 10.1128/jvi.01389-23

    KSHV RTA interacts with the host E3 ubiquitin ligase complex RNF20/40. (A) FLAG immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (B) RNF20 or RNF40 immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (C) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA (3xF-RTA), and the samples were subjected to immunoblot analysis. (D) 3xFLAG-tagged RTA, RNF20, and RNF40 were translated with TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI). The expressed proteins were then subjected to immunoprecipitation and immunoblot analysis. (E) In situ PLA using proximity probes against RTA and RNF20 was performed to visualize their interaction in iBCBL1-3xFLAG-RTA cells reactivated for 12 h. (F) Model representation of the full-length (FL) KSHV RTA and its truncation mutants. RING, RING-like domain; LR, leucine heptapeptide repeat; TAD, transactivation domain; and NLS, nuclear localization signal. (G) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA full-length (FL) and truncation mutants, and the samples were subjected to immunoblot analysis.
    Figure Legend Snippet: KSHV RTA interacts with the host E3 ubiquitin ligase complex RNF20/40. (A) FLAG immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (B) RNF20 or RNF40 immunoprecipitation was performed using iBCBL1-3xFLAG-RTA cells that were reactivated using Dox for 12 h, and the samples were subjected to immunoblot analysis. (C) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA (3xF-RTA), and the samples were subjected to immunoblot analysis. (D) 3xFLAG-tagged RTA, RNF20, and RNF40 were translated with TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI). The expressed proteins were then subjected to immunoprecipitation and immunoblot analysis. (E) In situ PLA using proximity probes against RTA and RNF20 was performed to visualize their interaction in iBCBL1-3xFLAG-RTA cells reactivated for 12 h. (F) Model representation of the full-length (FL) KSHV RTA and its truncation mutants. RING, RING-like domain; LR, leucine heptapeptide repeat; TAD, transactivation domain; and NLS, nuclear localization signal. (G) FLAG immunoprecipitation was performed using 293T cells transfected with 3xFLAG-tagged RTA full-length (FL) and truncation mutants, and the samples were subjected to immunoblot analysis.

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, In Situ

    RNF20 promotes KSHV lytic cycle in iBCBL1-3xFLAG-RTA cell line. (A) Immunoblot analysis of RNF20/40 and viral proteins in cells transduced with lenti-shControl or lenti-shRNF20 for 2 days and then reactivated for various time points. E: early viral protein and L: late viral protein. (B) MTT assay was performed to measure cell viability of cells transduced with lenti-shControl or lenti-shRNF20 at 3 dpi. (C) Viral gene expression measured by RT-qPCR at various time points. The fold change is calculated relative to shControl 0 hpi. IE: immediate early genes; E: early genes; and L: late genes. (D) Viral DNA level at various time points measured by qPCR. The fold change is calculated relative to shControl 0 hpi. (E) KSHV production was determined by transferring the virus-containing supernatant from reactivated lenti-shRNA transduced iBCBL1-3xFLAG-RTA to 293T cells. Total DNA in infected 293T cells was analyzed at 2 hpi. The amount of viral DNA was quantified by qPCR and normalized to host DNA. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 and n.s.: not significant (sample of n = 3).
    Figure Legend Snippet: RNF20 promotes KSHV lytic cycle in iBCBL1-3xFLAG-RTA cell line. (A) Immunoblot analysis of RNF20/40 and viral proteins in cells transduced with lenti-shControl or lenti-shRNF20 for 2 days and then reactivated for various time points. E: early viral protein and L: late viral protein. (B) MTT assay was performed to measure cell viability of cells transduced with lenti-shControl or lenti-shRNF20 at 3 dpi. (C) Viral gene expression measured by RT-qPCR at various time points. The fold change is calculated relative to shControl 0 hpi. IE: immediate early genes; E: early genes; and L: late genes. (D) Viral DNA level at various time points measured by qPCR. The fold change is calculated relative to shControl 0 hpi. (E) KSHV production was determined by transferring the virus-containing supernatant from reactivated lenti-shRNA transduced iBCBL1-3xFLAG-RTA to 293T cells. Total DNA in infected 293T cells was analyzed at 2 hpi. The amount of viral DNA was quantified by qPCR and normalized to host DNA. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 and n.s.: not significant (sample of n = 3).

    Techniques Used: Western Blot, Transduction, MTT Assay, Expressing, Quantitative RT-PCR, Transferring, Virus, shRNA, Infection

    RNF20 is necessary for inducing RTA expression during lytic KSHV reactivation. (A) Immunoblot analysis of RNF20/40 and viral proteins in BCBL1 cells transduced with lenti-shControl or lenti-shRNF20 for 2 days and then reactivated with TPA for various time points. IE: immediate early protein; E: early protein; and L: late protein. (B) Viral gene expression calculated relative to shControl 0 hpi was measured by RT-qPCR at various time points. IE: immediate early genes; E: early genes; and L: late genes. The degree of statistical significance in the reduction of lytic gene expression by shRNF20 was calculated by t-tests (*P ≤ 0.05 and n = 3).
    Figure Legend Snippet: RNF20 is necessary for inducing RTA expression during lytic KSHV reactivation. (A) Immunoblot analysis of RNF20/40 and viral proteins in BCBL1 cells transduced with lenti-shControl or lenti-shRNF20 for 2 days and then reactivated with TPA for various time points. IE: immediate early protein; E: early protein; and L: late protein. (B) Viral gene expression calculated relative to shControl 0 hpi was measured by RT-qPCR at various time points. IE: immediate early genes; E: early genes; and L: late genes. The degree of statistical significance in the reduction of lytic gene expression by shRNF20 was calculated by t-tests (*P ≤ 0.05 and n = 3).

    Techniques Used: Expressing, Western Blot, Transduction, Quantitative RT-PCR

    KSHV RTA utilizes RNF20/40 to induce cellular gene expression. (A) Host gene expression measured by RT-qPCR at 12 hpi using the iBCBL1-3xFLAG-RTA samples shown in Fig. 3. (B) Immunoblot analysis of RNF20 and KSHV RTA in iSLK cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then Dox was added to induce RTA expression. (C) MTT assay was performed to measure cell viability of cells transduced with lenti-shControl or lenti-shRNF20 at 3 dpi. (D) Host gene expression measured by RT-qPCR at 12 hpi. The degree of statistical significance in the reduction of host gene expression by shRNF20 was calculated by t-tests (*P ≤ 0.05, n.s.: not significant, and n = 3).
    Figure Legend Snippet: KSHV RTA utilizes RNF20/40 to induce cellular gene expression. (A) Host gene expression measured by RT-qPCR at 12 hpi using the iBCBL1-3xFLAG-RTA samples shown in Fig. 3. (B) Immunoblot analysis of RNF20 and KSHV RTA in iSLK cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then Dox was added to induce RTA expression. (C) MTT assay was performed to measure cell viability of cells transduced with lenti-shControl or lenti-shRNF20 at 3 dpi. (D) Host gene expression measured by RT-qPCR at 12 hpi. The degree of statistical significance in the reduction of host gene expression by shRNF20 was calculated by t-tests (*P ≤ 0.05, n.s.: not significant, and n = 3).

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transduction, MTT Assay

    KSHV RTA uses RNF20/40 to induce KSHV lytic promoters. (A) 293T cells were transfected with a KSHV ORF50 promoter luciferase reporter plasmid along with RTA, RNF20, and RNF40. Luciferase activity was measured, and the results were normalized to vector control. (B) 293T cells were transfected with a CMV promoter luciferase reporter plasmid along with RTA, RNF20, and RNF40. Luciferase activity was measured, and the results were normalized to vector control. (C) 293T cells were transfected with a luciferase reporter plasmid containing a KSHV promoter (ORF50, ORF57, or K2) along with RNF20/40 and an RTA WT or mutant. Luciferase activity was measured and normalized to vector control. (D) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV ORF50 promoter luciferase reporter plasmid along with KSHV RTA. (E) Luciferase activity of samples described in panel (D). Results were normalized to shControl vector. (F) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV ORF57 promoter luciferase reporter plasmid along with KSHV RTA. (G) Luciferase activity of samples described in panel (F). Results normalized to shControl vector. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).
    Figure Legend Snippet: KSHV RTA uses RNF20/40 to induce KSHV lytic promoters. (A) 293T cells were transfected with a KSHV ORF50 promoter luciferase reporter plasmid along with RTA, RNF20, and RNF40. Luciferase activity was measured, and the results were normalized to vector control. (B) 293T cells were transfected with a CMV promoter luciferase reporter plasmid along with RTA, RNF20, and RNF40. Luciferase activity was measured, and the results were normalized to vector control. (C) 293T cells were transfected with a luciferase reporter plasmid containing a KSHV promoter (ORF50, ORF57, or K2) along with RNF20/40 and an RTA WT or mutant. Luciferase activity was measured and normalized to vector control. (D) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV ORF50 promoter luciferase reporter plasmid along with KSHV RTA. (E) Luciferase activity of samples described in panel (D). Results were normalized to shControl vector. (F) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV ORF57 promoter luciferase reporter plasmid along with KSHV RTA. (G) Luciferase activity of samples described in panel (F). Results normalized to shControl vector. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).

    Techniques Used: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Mutagenesis, Western Blot, Transduction

    RTA binding to its target promoters is necessary for the synergistic promoter induction by RTA and RNF20/40. (A) 293T cells were transfected with a luciferase reporter plasmid containing different regions of the KSHV ORF50 promoter along with RTA with or without RNF20/40. Luciferase activity was measured, and the results were normalized to vector control. (B) Schematic of promoter luciferase reporters. WT and a mutant RTA response element (RRE) derived from ORF57 promoter were cloned into the luciferase reporter plasmid pGL4.27 containing a minimal promoter, which has only a TATA-box promoter element. (C) Luciferase activity of 293T cells co-transfected with RRE WT or mutant luciferase reporter plasmid with WT or mutant RTAs. Results were normalized to vector-transfected cells. (D) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV RRE luciferase reporter plasmid along with KSHV RTA. (E) Luciferase activity of samples described in panel (D). Results were normalized to shControl sample. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).
    Figure Legend Snippet: RTA binding to its target promoters is necessary for the synergistic promoter induction by RTA and RNF20/40. (A) 293T cells were transfected with a luciferase reporter plasmid containing different regions of the KSHV ORF50 promoter along with RTA with or without RNF20/40. Luciferase activity was measured, and the results were normalized to vector control. (B) Schematic of promoter luciferase reporters. WT and a mutant RTA response element (RRE) derived from ORF57 promoter were cloned into the luciferase reporter plasmid pGL4.27 containing a minimal promoter, which has only a TATA-box promoter element. (C) Luciferase activity of 293T cells co-transfected with RRE WT or mutant luciferase reporter plasmid with WT or mutant RTAs. Results were normalized to vector-transfected cells. (D) Immunoblot analysis of 293T cells transduced with lenti-shControl or lenti-shRNF20 for 2 days, then transfected with KSHV RRE luciferase reporter plasmid along with KSHV RTA. (E) Luciferase activity of samples described in panel (D). Results were normalized to shControl sample. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).

    Techniques Used: Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Mutagenesis, Derivative Assay, Clone Assay, Western Blot, Transduction

    Overexpression of RNF20/40 further promotes RTA-induced lytic viral gene expression. (A) Immunoblot analysis of 293TBAC16 cells co-transfected with KSHV RTA and RNF20/40 WT or RING domain deletion mutant. (B) Viral gene expression was measured by RT-qPCR. The degree of statistical significance is calculated by t-tests (*P ≤ 0.05 and n = 3).
    Figure Legend Snippet: Overexpression of RNF20/40 further promotes RTA-induced lytic viral gene expression. (A) Immunoblot analysis of 293TBAC16 cells co-transfected with KSHV RTA and RNF20/40 WT or RING domain deletion mutant. (B) Viral gene expression was measured by RT-qPCR. The degree of statistical significance is calculated by t-tests (*P ≤ 0.05 and n = 3).

    Techniques Used: Over Expression, Expressing, Western Blot, Transfection, Mutagenesis, Quantitative RT-PCR

    RNF20 is recruited to RTA binding sites on viral and host genomes during lytic reactivation of KSHV. (A) Co-localization of RNF20 with LANA-marked KSHV episomes was determined in iSLKBAC16-3xFLAG-LANA cells using immunofluorescence analysis of RNF20 and FLAG (LANA) during latency (0 hpi) and lytic reactivation (24 hpi). DAPI: nucleus; green: RNF20; and red: LANA. (B) Representative LANA puncta were connected by yellow line, and the co-localization of LANA with KDM2B along the line was analyzed by ImageJ. (C) CUT&RUN assay was performed on iBCBL1-3xFLAG RTA cells reactivated for 12 h to examine RTA and RNF20 binding to viral and host gene promoters.
    Figure Legend Snippet: RNF20 is recruited to RTA binding sites on viral and host genomes during lytic reactivation of KSHV. (A) Co-localization of RNF20 with LANA-marked KSHV episomes was determined in iSLKBAC16-3xFLAG-LANA cells using immunofluorescence analysis of RNF20 and FLAG (LANA) during latency (0 hpi) and lytic reactivation (24 hpi). DAPI: nucleus; green: RNF20; and red: LANA. (B) Representative LANA puncta were connected by yellow line, and the co-localization of LANA with KDM2B along the line was analyzed by ImageJ. (C) CUT&RUN assay was performed on iBCBL1-3xFLAG RTA cells reactivated for 12 h to examine RTA and RNF20 binding to viral and host gene promoters.

    Techniques Used: Binding Assay, Immunofluorescence

    RNF20 regulates RNA polymerase II occupancy on RTA promoter during lytic reactivation of KSHV. (A–D) iBCBL1-3xFLAG-RTA cells were transduced with lenti-shControl or lenti-shRNF20 then reactivated for 12 h, and ChIP-qPCR analysis was performed at the indicated sites of the RTA promoter and gene body. (A) H2BK120ub ChIP. (B) H3K4me3 ChIP. (C) RNA polymerase II (RNAP II) ChIP. (D) ChIP for RNAP II Serine 2 phosphorylation. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).
    Figure Legend Snippet: RNF20 regulates RNA polymerase II occupancy on RTA promoter during lytic reactivation of KSHV. (A–D) iBCBL1-3xFLAG-RTA cells were transduced with lenti-shControl or lenti-shRNF20 then reactivated for 12 h, and ChIP-qPCR analysis was performed at the indicated sites of the RTA promoter and gene body. (A) H2BK120ub ChIP. (B) H3K4me3 ChIP. (C) RNA polymerase II (RNAP II) ChIP. (D) ChIP for RNAP II Serine 2 phosphorylation. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).

    Techniques Used: Transduction

    Working model for RTA re-tasking RNF20/40 to promote the KSHV lytic cycle. Upon KSHV lytic reactivation, once RTA is expressed, RTA binds to its own gene promoter via interacting with RBPJk and recruits RNF20/40 to help autoregulate and sustain RTA expression. Our results suggest that RNF20/40 facilitates the recruitment of transcriptionally active RNAP II onto RTA gene resulting in increased viral gene expression. RTA binds to its viral and host target promoters, directly or indirectly, and uses RNF20/40 to promote the expression of its viral and host target genes as well. Created with BioRender.com.
    Figure Legend Snippet: Working model for RTA re-tasking RNF20/40 to promote the KSHV lytic cycle. Upon KSHV lytic reactivation, once RTA is expressed, RTA binds to its own gene promoter via interacting with RBPJk and recruits RNF20/40 to help autoregulate and sustain RTA expression. Our results suggest that RNF20/40 facilitates the recruitment of transcriptionally active RNAP II onto RTA gene resulting in increased viral gene expression. RTA binds to its viral and host target promoters, directly or indirectly, and uses RNF20/40 to promote the expression of its viral and host target genes as well. Created with BioRender.com.

    Techniques Used: Expressing

    immunoblotting with anti rnf20  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Cell Signaling Technology Inc immunoblotting with anti rnf20
    Immunoblotting With Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoblotting with anti rnf20/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    immunoblotting with anti rnf20 - by Bioz Stars, 2024-06
    86/100 stars

    Images

    immunoblotting with anti rnf20  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc immunoblotting with anti rnf20
    Immunoblotting With Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoblotting with anti rnf20/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    immunoblotting with anti rnf20 - by Bioz Stars, 2024-06
    94/100 stars

    Images

    apoe amino acids 141 160  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc apoe amino acids 141 160
    Apoe Amino Acids 141 160, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoe amino acids 141 160/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    apoe amino acids 141 160 - by Bioz Stars, 2024-06
    94/100 stars

    Images

    9425s rrid ab 2797700  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Cell Signaling Technology Inc 9425s rrid ab 2797700

    9425s Rrid Ab 2797700, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/9425s rrid ab 2797700/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    9425s rrid ab 2797700 - by Bioz Stars, 2024-06
    93/100 stars

    Images

    1) Product Images from "Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions"

    Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

    Journal: eLife

    doi: 10.7554/eLife.73524


    Figure Legend Snippet:

    Techniques Used: Software

    anti rnf20  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Cell Signaling Technology Inc anti rnf20

    Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rnf20/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rnf20 - by Bioz Stars, 2024-06
    93/100 stars

    Images

    1) Product Images from "Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions"

    Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

    Journal: eLife

    doi: 10.7554/eLife.73524


    Figure Legend Snippet:

    Techniques Used: Software

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Cell Signaling Technology Inc anti rnf20
    Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rnf20/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rnf20 - by Bioz Stars, 2024-06
    86/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc immunoblotting with anti rnf20
    Immunoblotting With Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoblotting with anti rnf20/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    immunoblotting with anti rnf20 - by Bioz Stars, 2024-06
    86/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc apoe amino acids 141 160
    Apoe Amino Acids 141 160, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoe amino acids 141 160/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    apoe amino acids 141 160 - by Bioz Stars, 2024-06
    94/100 stars
      Buy from Supplier

    93
    Cell Signaling Technology Inc 9425s rrid ab 2797700

    9425s Rrid Ab 2797700, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/9425s rrid ab 2797700/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    9425s rrid ab 2797700 - by Bioz Stars, 2024-06
    93/100 stars
      Buy from Supplier

    Image Search Results


    Journal: eLife

    Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

    doi: 10.7554/eLife.73524

    Figure Lengend Snippet:

    Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

    Techniques: Software