Journal: The Journal of Biological Chemistry

Article Title: The p90 ribosomal S6 kinase–UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor–associated factor 3

doi: 10.1074/jbc.M117.785170

Figure Lengend Snippet: UBR5 negatively regulates TRAF3–TBK1 signaling by a DUBA-independent mechanism. A, immunoblot analysis using whole-cell lysate of control or UBR5 shRNA stable HeLa cell lines transfected with control or DUBA siRNA for 48 h. pTBK1 indicates the phospho-Ser172 TBK1 antibody. All of the immunoblots were completed using the same cell lysates. Data are representatives of three independent experiments. B, the Dual-Luciferase assay for the IFNβ gene promoter were completed from the HeLa cell lysates 72 h after transfection with control (siCon) or UBR5 siRNA (siUBR5) as described under “Experimental procedures.” Data are shown as the mean ± S.D. of four samples from a representative experiment performed three times. C, qPCR analysis of the Ifnb1 mRNA level in control or UBR5 siRNA–transfected HeLa cells. Data are shown as the mean ± S.D. of four samples from a representative experiment performed three times.

Article Snippet: siRNA against Ago1 (siRNA no.1046447), Ago2 (siRNA no.1046457), TNRC6A (siRNA no.1154883), and negative control siRNA (SN-1003) were obtained from Bioneer (Daejeon, Republic of Korea). siRNA against DUBA (L-013823-00) was obtained from Dharmacon. siRNA against UBR5 was obtained from Santa Cruz Biotechnology (sc-43744) and Bioneer (siRNA no. 1045520). siRNA against RSK1, -2, and -3 were obtained from Dharmacon (L-003025-00, L-003026, and L-004663-00) and Bioneer (siRNA no.1131587, 1131617, and 1131598).

Techniques: Western Blot, Control, shRNA, Transfection, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: The p90 ribosomal S6 kinase–UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor–associated factor 3

doi: 10.1074/jbc.M117.785170

Figure Lengend Snippet: UBR5 regulates TRAF3 expression through miRNA-mediated translational repression. A and B, immunoblot analysis of the indicated proteins using the whole-cell lysates of HeLa cell lines stably expressing control or UBR5 shRNA treated with DMSO or MG132 (10 μm) for 6 h (A) or with chloroquine (100 nm) for the indicated time periods (B). C and D, immunoblot analysis of the indicated proteins using the whole-cell lysates of HeLa cells transfected with siRNAs against Ago1/2, GW182, UBR5, or control siRNA for 72 h. E, luciferase assay of TRAF3 3′-UTR luciferase activity using lysate of HeLa cells that were transfected with siRNAs against UBR5, Ago1 or -2, or control siRNA. Data are shown as the mean ± S.D. of duplicate samples from a representative experiment performed three times. F, qPCR analysis of UBR5 and TRAF3 mRNA level in control and UBR5 siRNA–transfected HeLa cells. Data are shown as the mean ± S.D. of duplicate samples from a representative experiment performed three times (n.s. (non-significant), Student's t test). G, immunoblot analysis of exogenous Myc-tagged TRAF3 containing the 3′-UTR region (Myc–TRAF3U) or not (Myc–TRAF3) in HeLa cells that were transfected with control or UBR5 siRNA for 72 h. Data are representative of at least two independent experiments.

Article Snippet: siRNA against Ago1 (siRNA no.1046447), Ago2 (siRNA no.1046457), TNRC6A (siRNA no.1154883), and negative control siRNA (SN-1003) were obtained from Bioneer (Daejeon, Republic of Korea). siRNA against DUBA (L-013823-00) was obtained from Dharmacon. siRNA against UBR5 was obtained from Santa Cruz Biotechnology (sc-43744) and Bioneer (siRNA no. 1045520). siRNA against RSK1, -2, and -3 were obtained from Dharmacon (L-003025-00, L-003026, and L-004663-00) and Bioneer (siRNA no.1131587, 1131617, and 1131598).

Techniques: Expressing, Western Blot, Stable Transfection, Control, shRNA, Transfection, Luciferase, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The p90 ribosomal S6 kinase–UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor–associated factor 3

doi: 10.1074/jbc.M117.785170

Figure Lengend Snippet: p90RSK phosphorylates UBR5 at multiple sites. A, conservation of RXRXXS motif of UBR5 Ser2483 among various species. B, FLAG–wild-type UBR5 (WT) or the indicated UBR5 mutant (S2483A (SA) and S2483E (SE)) were transfected in COS-1 cells. The FLAG–UBR5 proteins were isolated by immunoprecipitation and subjected to immunoblot analysis for phosphorylation using the indicated antibodies (top and middle panels). The cell lysates were subjected to direct immunoblot (bottom panel, Input = 2% of the total lysates). C, after 24-h serum starvation, the HeLa cells transfected with FLAG–UBR5 were stimulated with EGF (100 ng/ml), PMA (50 ng/ml), or FBS (20%) for 15 min. The lysates were subjected to immunoprecipitation followed by immunoblot analysis using the indicated antibodies. D, after 24-h serum starvation, the COS-1 cells were pretreated with GDC0349 (1 μm) or BI-D1870 (10 μm) for 30 min prior to EGF (100 ng/ml) stimulation for 15 min. The FLAG–UBR5 proteins were isolated by immunoprecipitation and subjected to immunoblot analysis using the indicated antibodies. E, the serum-starved COS-1 cells were stimulated with EGF for 15 min in the absence or presence of BI-D1870 (for 30 min). The cell lysates were subjected to immunoblot analysis using the indicated antibodies. F, COS-1 cells were transfected with control siRNA or p90RSK1, -2, or -3 siRNA and empty vector or FLAG–UBR5 as indicated. The FLAG–UBR5 proteins were isolated by immunoprecipitation and subjected to immunoblot analysis using the indicated antibodies (IP), and the cell lysates were subjected to direct immunoblot with the indicated antibodies (Input). G, COS-1 cells were cotransfected with empty vector or Myc–avian p90RSK and empty vector or FLAG–UBR5 as indicated. The FLAG–UBR5 proteins were isolated by immunoprecipitation and subjected to immunoblot analysis using indicated antibodies (IP), and the cell lysates were subjected to direct immunoblot with the indicated antibodies (Input). H, COS-1 cells were cotransfected with empty vector or HA–human p90RSK1 (hRSK1) and empty vector or FLAG–UBR5 as indicated. The FLAG–UBR5 proteins were isolated by immunoprecipitation and subjected to immunoblot analysis with the indicated antibodies to detect UBR5–p90RSK1 interaction (IP), and the cell lysates were subjected to direct immunoblot with the same antibodies (Input). I–K, in vitro kinase assay using FLAG–UBR5 and Myc–avian p90RSK (I), FLAG-WT-UBR5, FLAG-T637A-UBR5, FLAG-S2483A-UBR5, and Myc–avian p90RSK (J), FLAG-WT-UBR5, FLAG-S2483A-UBR5 (1A), T637/S1227/S2483A-UBR5 (3A), and Myc–avian p90RSK (K) as described under “Experimental procedures.” CBS, Coomassie Blue staining.

Article Snippet: siRNA against Ago1 (siRNA no.1046447), Ago2 (siRNA no.1046457), TNRC6A (siRNA no.1154883), and negative control siRNA (SN-1003) were obtained from Bioneer (Daejeon, Republic of Korea). siRNA against DUBA (L-013823-00) was obtained from Dharmacon. siRNA against UBR5 was obtained from Santa Cruz Biotechnology (sc-43744) and Bioneer (siRNA no. 1045520). siRNA against RSK1, -2, and -3 were obtained from Dharmacon (L-003025-00, L-003026, and L-004663-00) and Bioneer (siRNA no.1131587, 1131617, and 1131598).

Techniques: Mutagenesis, Transfection, Isolation, Immunoprecipitation, Western Blot, Phospho-proteomics, Control, Plasmid Preparation, In Vitro, Kinase Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: The p90 ribosomal S6 kinase–UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor–associated factor 3

doi: 10.1074/jbc.M117.785170

Figure Lengend Snippet: p90RSK negatively regulates TRAF3–TBK1signaling via UBR5. A and B, immunoblot analysis of the indicated proteins using the whole-cell lysates of HeLa cells that were treated with DMSO or BI-D1870 for 12 h (A) or transfected with siRNAs against p90RSK1/2/3 and control siRNA for 48 h (B). All of the immunoblots were completed using the same cell lysates. C, qPCR analysis of Ifnb1 mRNA level using HeLa cells treated with DMSO or BI-D1870 for 12 h. Data are shown as the mean ± S.D. of duplicate samples from a representative experiment performed three times. D, Dual-Luciferase assays were performed using a TRAF3 3′-UTR reporter in HeLa cells transfected with siRNAs against p90RSK1/2/3, Ago-1 and -2, and control siRNA. Data are shown as the mean ± S.D. of duplicate samples from a representative experiment performed three times. E, HeLa cells were cotransfected with empty vector or Myc-TRAF3U and empty vector or HA–human RSK CA as indicated for 48 h. After a 12-h serum starvation, the cell lysates were subjected to immunoblot analysis with the indicated antibodies. F, immunoblot analysis of indicated proteins using DMSO or BI-D1870 (for 12 h) treated HeLa cells transfected with siRNAs against control or UBR5 for 48 h. G, densitometry analysis of immunoblot bands from F from four independent experiments. TRAF3 bands were normalized to tubulin bands. Data are shown as the mean ± S.D. from four independent experiments (*, p = 0.005; **, p = 0.0005; ***, p = 0.002; and n.s. (non-significant), Student's t test).

Article Snippet: siRNA against Ago1 (siRNA no.1046447), Ago2 (siRNA no.1046457), TNRC6A (siRNA no.1154883), and negative control siRNA (SN-1003) were obtained from Bioneer (Daejeon, Republic of Korea). siRNA against DUBA (L-013823-00) was obtained from Dharmacon. siRNA against UBR5 was obtained from Santa Cruz Biotechnology (sc-43744) and Bioneer (siRNA no. 1045520). siRNA against RSK1, -2, and -3 were obtained from Dharmacon (L-003025-00, L-003026, and L-004663-00) and Bioneer (siRNA no.1131587, 1131617, and 1131598).

Techniques: Western Blot, Transfection, Control, Luciferase, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The p90 ribosomal S6 kinase–UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor–associated factor 3

doi: 10.1074/jbc.M117.785170

Figure Lengend Snippet: p90RSK–UBR5 pathway regulates KRAS and p60 katanin expression. A, Dual-Luciferase assays using a KRAS 3′-UTR reporter were carried out using the whole-cell lysates of control or UBR5 shRNA stable HeLa cells. Data are shown as the mean ± S.D. of duplicate samples from a representative experiment performed three times. B–D, KRAS 3′-UTR reporter assays were performed using the cells treated with DMSO or BI-D1870 for 12 h (B), transfected with control or p90RSK1, -2, and -3 siRNAs for 48 h (C), or transfected with empty vector or HA–RSK CA for 48 h (D). Data are shown as the mean ± S.D. of duplicate samples from a representative experiment performed two times. E, HEK293 cells were cotransfected with empty vector (−) or FLAG–wild type (+), 3E, or 3A mutant UBR5 and Myc–GFP–GW182 as indicated. After 48 h, the Myc–GFP–GW182 cells were immunoprecipitated using anti-Myc antibody and subjected to immunoblot analysis with the indicated antibodies to detect UBR5 and GW182 interaction (IP), and the cell lysates were subjected to direct immunoblot with same antibodies (Input). F, HEK293 cells were cotransfected with empty vector (−) or FLAG–wild type(+), 3E, or 3A mutant UBR5 and HA–Ago2 as indicated. After 48 h, the FLAG–UBR5 proteins were immunoprecipitated using anti-FLAG antibody and subjected to immunoblot analysis with the indicated antibodies to detect UBR5 and Ago2 interaction (IP), and the cell lysates were subjected to direct immunoblot with same antibodies (Input). G, luciferase assay of KRAS 3′-UTR luciferase activity using control or UBR5 shRNA stable HEK293 cell lysates cotransfected with Luc–KRAS 3′-UTR, pRL-CMV, and empty vector (−), FLAG–WT UBR5, 3E, or 3A mutant. Data are shown as the mean ± S.D. of duplicate samples from a representative experiment performed twice. (*, p = 0.03; **, p = 0.0006; ***, p = 0.005; Student's t test).

Article Snippet: siRNA against Ago1 (siRNA no.1046447), Ago2 (siRNA no.1046457), TNRC6A (siRNA no.1154883), and negative control siRNA (SN-1003) were obtained from Bioneer (Daejeon, Republic of Korea). siRNA against DUBA (L-013823-00) was obtained from Dharmacon. siRNA against UBR5 was obtained from Santa Cruz Biotechnology (sc-43744) and Bioneer (siRNA no. 1045520). siRNA against RSK1, -2, and -3 were obtained from Dharmacon (L-003025-00, L-003026, and L-004663-00) and Bioneer (siRNA no.1131587, 1131617, and 1131598).

Techniques: Expressing, Luciferase, Control, shRNA, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Western Blot, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The p90 ribosomal S6 kinase–UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor–associated factor 3

doi: 10.1074/jbc.M117.785170

Figure Lengend Snippet: Schematic overview of p90RSK-mediated regulation of UBR5-containing miRISC.

Article Snippet: siRNA against Ago1 (siRNA no.1046447), Ago2 (siRNA no.1046457), TNRC6A (siRNA no.1154883), and negative control siRNA (SN-1003) were obtained from Bioneer (Daejeon, Republic of Korea). siRNA against DUBA (L-013823-00) was obtained from Dharmacon. siRNA against UBR5 was obtained from Santa Cruz Biotechnology (sc-43744) and Bioneer (siRNA no. 1045520). siRNA against RSK1, -2, and -3 were obtained from Dharmacon (L-003025-00, L-003026, and L-004663-00) and Bioneer (siRNA no.1131587, 1131617, and 1131598).

Techniques:

Journal: American Journal of Cancer Research

Article Title: Prolactin-inducible EDD E3 ubiquitin ligase promotes TORC1 signalling, anti-apoptotic protein expression, and drug resistance in breast cancer cells

doi:

Figure Lengend Snippet: Loss of EDD decreases drug resistance in MCF-7 cells. MCF-7 cells were infected with shRNA lentiviral particles to knockdown EDD and puromycin-resistant clones were selected. In (A), Western analysis showed EDD gene knockdown in clone 1.1C but not in clone 1.1A. Each clone was treated with (B) 0.3125 μM cisplatin, (C) 0.023 μM doxorubicin, (D) 16.59 μM rapamycin or (E) 20 μM tamoxifen for up to 5 days. On Day 1-5, viable cell counts ± drug treatment were measured (left panels). Day 4 and 5 cell counts were replotted to clearly show statistical significance (middle panels) or expressed as % cell survival of drug-treated clone 1.1A or 1.1C, each compared to its untreated control (right panels). Mean ± SEM (n = 3-4). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Generation of shEDD-MCF-7 clones MCF-7 cells were stably transduced with shRNA targeting EDD through infection with lentiviral particles (sc-43744-V, Santa Cruz Biotechnology), following the manufacturer’s protocol.

Techniques: Infection, shRNA, Knockdown, Clone Assay, Western Blot, Control