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ruvbl2 (1:2500)  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ruvbl2 (1:2500)
    Identification of RUVBL1 as a factor required for Nrf1 transcriptional activity. A, schematic of the reporter construct used in the cell-based screening system. This lentiviral reporter construct expressed firefly luciferase under the control of 8xARE upstream of a minimal promoter (Pmin), along with Renilla luciferase driven by the hPGK promoter. B, NIH-3T3 cells with stable incorporation of the reporter system described above and that were either wild-type (WT 8xARE-Luc) or Nrf1-deficient (Nrf1−/− 8xARE-Luc) were treated with DMSO or 200 nm CFZ overnight and analyzed by immunoblotting using antibodies specific for Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown. C, WT 8xARE-Luc and Nrf1−/− 8xARE-Luc cells were treated for 16 h with increasing concentrations of CFZ (0, 20, 50, 100, 150, and 200 nm) and then subjected to Dual-Luciferase assays to measure the firefly and Renilla luciferase activity values. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). D, WT 8xARE-Luc cells were treated with 200 nm CFZ alone or in combination with 10 μm NMS-873 and compared with the DMSO-treated control for 16 h. The cell lysates were then used for luciferase assays. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). E, WT 8xARE-Luc cells were transfected with a focused library of siRNAs targeting several epigenetic factors and other candidate genes. Forty-eight hours after transfection, the cells were further treated with 200 nm CFZ overnight and assayed for luciferase activity. Error bars denote S.D. (n = 3). F, WT NIH-3T3 cells were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1 and further treated with 200 nm CFZ, as indicated, for 8 h. RNA extracted from these cells was then subjected to quantitative RT-PCR with primers specific for representative proteasome subunit genes as shown. The transcript levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). G, NIH-3T3 cells treated as described in F were used for immunoblotting with antibodies against Nrf1, RUVBL1, <t>RUVBL2,</t> ubiquitin, and β-actin as indicated. The experiments were performed three independent times, and a representative blot is shown.
    Ruvbl2 (1:2500), supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ruvbl2+(1%3A2500)/ruvbl2++1+2500++antibody/pmc06369275-308-10-20
    Average 90 stars, based on 1 article reviews
    ruvbl2 (1:2500) - by Bioz Stars, 2026-09
    90/100 stars

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    1) Product Images from "Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex"

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.006290

    Identification of RUVBL1 as a factor required for Nrf1 transcriptional activity. A, schematic of the reporter construct used in the cell-based screening system. This lentiviral reporter construct expressed firefly luciferase under the control of 8xARE upstream of a minimal promoter (Pmin), along with Renilla luciferase driven by the hPGK promoter. B, NIH-3T3 cells with stable incorporation of the reporter system described above and that were either wild-type (WT 8xARE-Luc) or Nrf1-deficient (Nrf1−/− 8xARE-Luc) were treated with DMSO or 200 nm CFZ overnight and analyzed by immunoblotting using antibodies specific for Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown. C, WT 8xARE-Luc and Nrf1−/− 8xARE-Luc cells were treated for 16 h with increasing concentrations of CFZ (0, 20, 50, 100, 150, and 200 nm) and then subjected to Dual-Luciferase assays to measure the firefly and Renilla luciferase activity values. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). D, WT 8xARE-Luc cells were treated with 200 nm CFZ alone or in combination with 10 μm NMS-873 and compared with the DMSO-treated control for 16 h. The cell lysates were then used for luciferase assays. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). E, WT 8xARE-Luc cells were transfected with a focused library of siRNAs targeting several epigenetic factors and other candidate genes. Forty-eight hours after transfection, the cells were further treated with 200 nm CFZ overnight and assayed for luciferase activity. Error bars denote S.D. (n = 3). F, WT NIH-3T3 cells were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1 and further treated with 200 nm CFZ, as indicated, for 8 h. RNA extracted from these cells was then subjected to quantitative RT-PCR with primers specific for representative proteasome subunit genes as shown. The transcript levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). G, NIH-3T3 cells treated as described in F were used for immunoblotting with antibodies against Nrf1, RUVBL1, RUVBL2, ubiquitin, and β-actin as indicated. The experiments were performed three independent times, and a representative blot is shown.
    Figure Legend Snippet: Identification of RUVBL1 as a factor required for Nrf1 transcriptional activity. A, schematic of the reporter construct used in the cell-based screening system. This lentiviral reporter construct expressed firefly luciferase under the control of 8xARE upstream of a minimal promoter (Pmin), along with Renilla luciferase driven by the hPGK promoter. B, NIH-3T3 cells with stable incorporation of the reporter system described above and that were either wild-type (WT 8xARE-Luc) or Nrf1-deficient (Nrf1−/− 8xARE-Luc) were treated with DMSO or 200 nm CFZ overnight and analyzed by immunoblotting using antibodies specific for Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown. C, WT 8xARE-Luc and Nrf1−/− 8xARE-Luc cells were treated for 16 h with increasing concentrations of CFZ (0, 20, 50, 100, 150, and 200 nm) and then subjected to Dual-Luciferase assays to measure the firefly and Renilla luciferase activity values. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). D, WT 8xARE-Luc cells were treated with 200 nm CFZ alone or in combination with 10 μm NMS-873 and compared with the DMSO-treated control for 16 h. The cell lysates were then used for luciferase assays. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). E, WT 8xARE-Luc cells were transfected with a focused library of siRNAs targeting several epigenetic factors and other candidate genes. Forty-eight hours after transfection, the cells were further treated with 200 nm CFZ overnight and assayed for luciferase activity. Error bars denote S.D. (n = 3). F, WT NIH-3T3 cells were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1 and further treated with 200 nm CFZ, as indicated, for 8 h. RNA extracted from these cells was then subjected to quantitative RT-PCR with primers specific for representative proteasome subunit genes as shown. The transcript levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). G, NIH-3T3 cells treated as described in F were used for immunoblotting with antibodies against Nrf1, RUVBL1, RUVBL2, ubiquitin, and β-actin as indicated. The experiments were performed three independent times, and a representative blot is shown.

    Techniques Used: Activity Assay, Construct, Luciferase, Western Blot, Transfection, Quantitative RT-PCR

    Depletion of RUVBL1 impairs the transcriptional function of Nrf1 in different cancer cell lines. A, the cell lines HCT116, MDA-MB-231, and MIA-PaCa2 were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1. Forty-eight hours after transfection, the cells were treated with 200 nm CFZ for 8 h and then analyzed by quantitative RT-PCR to measure representative proteasome subunit gene mRNA levels. The mRNA levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). B, the cell lines above were treated similarly as described in A and subjected to immunoblotting with antibodies specific for RUVBL1, RUVBL2, Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown.
    Figure Legend Snippet: Depletion of RUVBL1 impairs the transcriptional function of Nrf1 in different cancer cell lines. A, the cell lines HCT116, MDA-MB-231, and MIA-PaCa2 were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1. Forty-eight hours after transfection, the cells were treated with 200 nm CFZ for 8 h and then analyzed by quantitative RT-PCR to measure representative proteasome subunit gene mRNA levels. The mRNA levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). B, the cell lines above were treated similarly as described in A and subjected to immunoblotting with antibodies specific for RUVBL1, RUVBL2, Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown.

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot

    Nrf1 interacts with the TIP60 complex. A, WT and Nrf1−/− NIH-3T3 cell lines were treated with 200 nm CFZ for 8 h. The cells were then subjected to ChIP with IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for ARE-containing promoter regions of the proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote S.D. (n = 3). B, HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated with 200 nm CFZ for 8 h or left untreated. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times, and a representative blot is shown.
    Figure Legend Snippet: Nrf1 interacts with the TIP60 complex. A, WT and Nrf1−/− NIH-3T3 cell lines were treated with 200 nm CFZ for 8 h. The cells were then subjected to ChIP with IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for ARE-containing promoter regions of the proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote S.D. (n = 3). B, HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated with 200 nm CFZ for 8 h or left untreated. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times, and a representative blot is shown.

    Techniques Used: Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Immunoprecipitation, Western Blot

    Related Articles

    Activity Assay:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Construct:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Luciferase:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Western Blot:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Transfection:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Quantitative RT-PCR:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Real-time Polymerase Chain Reaction:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Stable Transfection:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Expressing:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Immunoprecipitation:

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).



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    ruvbl2 (1:2500)  (Cell Signaling Technology Inc)
    90
    Cell Signaling Technology Inc ruvbl2 (1:2500)
    Identification of RUVBL1 as a factor required for Nrf1 transcriptional activity. A, schematic of the reporter construct used in the cell-based screening system. This lentiviral reporter construct expressed firefly luciferase under the control of 8xARE upstream of a minimal promoter (Pmin), along with Renilla luciferase driven by the hPGK promoter. B, NIH-3T3 cells with stable incorporation of the reporter system described above and that were either wild-type (WT 8xARE-Luc) or Nrf1-deficient (Nrf1−/− 8xARE-Luc) were treated with DMSO or 200 nm CFZ overnight and analyzed by immunoblotting using antibodies specific for Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown. C, WT 8xARE-Luc and Nrf1−/− 8xARE-Luc cells were treated for 16 h with increasing concentrations of CFZ (0, 20, 50, 100, 150, and 200 nm) and then subjected to Dual-Luciferase assays to measure the firefly and Renilla luciferase activity values. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). D, WT 8xARE-Luc cells were treated with 200 nm CFZ alone or in combination with 10 μm NMS-873 and compared with the DMSO-treated control for 16 h. The cell lysates were then used for luciferase assays. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). E, WT 8xARE-Luc cells were transfected with a focused library of siRNAs targeting several epigenetic factors and other candidate genes. Forty-eight hours after transfection, the cells were further treated with 200 nm CFZ overnight and assayed for luciferase activity. Error bars denote S.D. (n = 3). F, WT NIH-3T3 cells were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1 and further treated with 200 nm CFZ, as indicated, for 8 h. RNA extracted from these cells was then subjected to quantitative RT-PCR with primers specific for representative proteasome subunit genes as shown. The transcript levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). G, NIH-3T3 cells treated as described in F were used for immunoblotting with antibodies against Nrf1, RUVBL1, <t>RUVBL2,</t> ubiquitin, and β-actin as indicated. The experiments were performed three independent times, and a representative blot is shown.
    Ruvbl2 (1:2500), supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ruvbl2+(1%3A2500)/ruvbl2++1+2500++antibody/pmc06369275-308-10-20
    Average 90 stars, based on 1 article reviews
    ruvbl2 (1:2500) - by Bioz Stars, 2026-09
    90/100 stars
    		  Buy from Supplier

    Image Search Results


    Identification of RUVBL1 as a factor required for Nrf1 transcriptional activity. A, schematic of the reporter construct used in the cell-based screening system. This lentiviral reporter construct expressed firefly luciferase under the control of 8xARE upstream of a minimal promoter (Pmin), along with Renilla luciferase driven by the hPGK promoter. B, NIH-3T3 cells with stable incorporation of the reporter system described above and that were either wild-type (WT 8xARE-Luc) or Nrf1-deficient (Nrf1−/− 8xARE-Luc) were treated with DMSO or 200 nm CFZ overnight and analyzed by immunoblotting using antibodies specific for Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown. C, WT 8xARE-Luc and Nrf1−/− 8xARE-Luc cells were treated for 16 h with increasing concentrations of CFZ (0, 20, 50, 100, 150, and 200 nm) and then subjected to Dual-Luciferase assays to measure the firefly and Renilla luciferase activity values. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). D, WT 8xARE-Luc cells were treated with 200 nm CFZ alone or in combination with 10 μm NMS-873 and compared with the DMSO-treated control for 16 h. The cell lysates were then used for luciferase assays. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). E, WT 8xARE-Luc cells were transfected with a focused library of siRNAs targeting several epigenetic factors and other candidate genes. Forty-eight hours after transfection, the cells were further treated with 200 nm CFZ overnight and assayed for luciferase activity. Error bars denote S.D. (n = 3). F, WT NIH-3T3 cells were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1 and further treated with 200 nm CFZ, as indicated, for 8 h. RNA extracted from these cells was then subjected to quantitative RT-PCR with primers specific for representative proteasome subunit genes as shown. The transcript levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). G, NIH-3T3 cells treated as described in F were used for immunoblotting with antibodies against Nrf1, RUVBL1, RUVBL2, ubiquitin, and β-actin as indicated. The experiments were performed three independent times, and a representative blot is shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

    doi: 10.1074/jbc.RA118.006290

    Figure Lengend Snippet: Identification of RUVBL1 as a factor required for Nrf1 transcriptional activity. A, schematic of the reporter construct used in the cell-based screening system. This lentiviral reporter construct expressed firefly luciferase under the control of 8xARE upstream of a minimal promoter (Pmin), along with Renilla luciferase driven by the hPGK promoter. B, NIH-3T3 cells with stable incorporation of the reporter system described above and that were either wild-type (WT 8xARE-Luc) or Nrf1-deficient (Nrf1−/− 8xARE-Luc) were treated with DMSO or 200 nm CFZ overnight and analyzed by immunoblotting using antibodies specific for Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown. C, WT 8xARE-Luc and Nrf1−/− 8xARE-Luc cells were treated for 16 h with increasing concentrations of CFZ (0, 20, 50, 100, 150, and 200 nm) and then subjected to Dual-Luciferase assays to measure the firefly and Renilla luciferase activity values. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). D, WT 8xARE-Luc cells were treated with 200 nm CFZ alone or in combination with 10 μm NMS-873 and compared with the DMSO-treated control for 16 h. The cell lysates were then used for luciferase assays. Normalized luciferase activity is shown. Error bars denote S.D. (n = 3). E, WT 8xARE-Luc cells were transfected with a focused library of siRNAs targeting several epigenetic factors and other candidate genes. Forty-eight hours after transfection, the cells were further treated with 200 nm CFZ overnight and assayed for luciferase activity. Error bars denote S.D. (n = 3). F, WT NIH-3T3 cells were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1 and further treated with 200 nm CFZ, as indicated, for 8 h. RNA extracted from these cells was then subjected to quantitative RT-PCR with primers specific for representative proteasome subunit genes as shown. The transcript levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). G, NIH-3T3 cells treated as described in F were used for immunoblotting with antibodies against Nrf1, RUVBL1, RUVBL2, ubiquitin, and β-actin as indicated. The experiments were performed three independent times, and a representative blot is shown.

    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Techniques: Activity Assay, Construct, Luciferase, Western Blot, Transfection, Quantitative RT-PCR

    Depletion of RUVBL1 impairs the transcriptional function of Nrf1 in different cancer cell lines. A, the cell lines HCT116, MDA-MB-231, and MIA-PaCa2 were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1. Forty-eight hours after transfection, the cells were treated with 200 nm CFZ for 8 h and then analyzed by quantitative RT-PCR to measure representative proteasome subunit gene mRNA levels. The mRNA levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). B, the cell lines above were treated similarly as described in A and subjected to immunoblotting with antibodies specific for RUVBL1, RUVBL2, Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

    doi: 10.1074/jbc.RA118.006290

    Figure Lengend Snippet: Depletion of RUVBL1 impairs the transcriptional function of Nrf1 in different cancer cell lines. A, the cell lines HCT116, MDA-MB-231, and MIA-PaCa2 were either control (Ctrl)-transfected or transfected with siRNAs targeting RUVBL1. Forty-eight hours after transfection, the cells were treated with 200 nm CFZ for 8 h and then analyzed by quantitative RT-PCR to measure representative proteasome subunit gene mRNA levels. The mRNA levels of 18S rRNA were used for normalization. Error bars denote S.D. (n = 3). B, the cell lines above were treated similarly as described in A and subjected to immunoblotting with antibodies specific for RUVBL1, RUVBL2, Nrf1, ubiquitin, and β-actin. The experiments were performed three independent times, and a representative blot is shown.

    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Techniques: Transfection, Quantitative RT-PCR, Western Blot

    Nrf1 interacts with the TIP60 complex. A, WT and Nrf1−/− NIH-3T3 cell lines were treated with 200 nm CFZ for 8 h. The cells were then subjected to ChIP with IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for ARE-containing promoter regions of the proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote S.D. (n = 3). B, HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated with 200 nm CFZ for 8 h or left untreated. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times, and a representative blot is shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

    doi: 10.1074/jbc.RA118.006290

    Figure Lengend Snippet: Nrf1 interacts with the TIP60 complex. A, WT and Nrf1−/− NIH-3T3 cell lines were treated with 200 nm CFZ for 8 h. The cells were then subjected to ChIP with IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for ARE-containing promoter regions of the proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote S.D. (n = 3). B, HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated with 200 nm CFZ for 8 h or left untreated. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times, and a representative blot is shown.

    Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), ubiquitin (1:3000), and cleaved caspase-3 (1:3000) (all from Cell Signaling Technology); INO80 (1:500, a gift from Dr. Landry ( 45 )); PIH1 (1:1000, Proteintech); and TIP60 (1:1500, Abcam) and β-actin (1:10,000, Sigma-Aldrich).

    Techniques: Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Immunoprecipitation, Western Blot