Journal: PLoS Pathogens

Article Title: Uridine Composition of the Poly-U/UC Tract of HCV RNA Defines Non-Self Recognition by RIG-I

doi: 10.1371/journal.ppat.1002839

Figure Lengend Snippet: A) Induction of the IFN-β-promoter in Huh7 cells transfected with equal moles of tRNA, full-length JFH1, JFH1 pU/UC, or Con1 pU/UC RNA. IFN-β-promoter luciferase activity is shown as mean IFN-β fold index (compared to cells with No RNA, ± s.d. for three replicates). Huh7 cells were transfected with the various RNA constructs and 16 hours later cells were harvested for dual luciferase activity. Asterisks indicate a significant difference compared to No RNA control as determined by a one-way ANOVA adjusted with Bonferroni's multiple comparison test (*P<0.05, **P<0.01, ***P<0.001). B) Induction of the IFN-β-promoter in Huh7 or Huh7.5 cells transfected with 350 ng of the indicated RNA constructs. IFN-β-promoter luciferase activity is shown as the mean IFN-β fold index ± s.d. for three replicates, and data was normalized to the No RNA control. Cells were harvested for dual luciferase activity 16 hours post-RNA transfection. Asterisks indicate a significant difference compared to No RNA control as determined by a one-way ANOVA adjusted with Bonferroni's multiple comparison test (*P<0.05, **P<0.001). C) The abundance of phospho-IRF-3 (Ser396), total IRF-3, RIG-I, ISG56, and tubulin were measured by immunoblot. Huh7 cells were transfected with the indicated RNA constructs and cells were harvested for protein analysis 16 hours later. RIG-I and ISG56 are IFN-β-stimulated genes. The ratio of phospho-IRF-3/total IRF-3 was calculated by measuring the relative immunoblot band intensities using ImageJ software (NIH). Data shown in all panels are representative of three independent experiments.

Article Snippet: RNA transfection was conducted in a 48-well plate format using the Trans IT-mRNA Transfection kit (Mirus) as per the manufacturer's instructions.

Techniques: Transfection, Luciferase, Activity Assay, Construct, Control, Comparison, Western Blot, Software

Journal: PLoS Pathogens

Article Title: Uridine Composition of the Poly-U/UC Tract of HCV RNA Defines Non-Self Recognition by RIG-I

doi: 10.1371/journal.ppat.1002839

Figure Lengend Snippet: A) Huh7 cells were transfected with the indicated poly-U/UC RNA constructs 12 hours prior to HCV infection (MOI = 0.1), and virus production was assessed 48 hours post-infection. Data shown are means ± s.d. for three replicates. Asterisks indicate a significant difference compared to No RNA control as determined by a one-way ANOVA adjusted with Bonferroni's multiple comparison test (*P<0.001, **P≤0.0001). B) Wild-type mice (n = 2) received 200 µg of X-region RNA, X-region-U34 RNA, Con1 pU/UC RNA, or Δcore RNA. Mock-transfected wild-type mice (n = 1) received PBS. Comparative measurements of hepatic mRNA and protein expression were measured 8 hours post-transfection. Real-time quantitative PCR was performed to examine expression of IFN-β , CCL5 , Ifit2 , ISG15 , and GAPDH . Results were normalized to the expression of mouse GAPDH mRNA, and mRNA fold index was normalized to Mock controls. Data shown are means ± s.d. for two replicates, and gene expression data was confirmed by two independent real-time PCR analyses. Asterisks indicate a significant difference as determined by a one-way ANOVA adjusted with Bonferroni's multiple comparison test (*P<0.05, **P<0.01, ***P<0.001). C) Following RNA transfection, mouse livers were recovered and immunohistochemistry staining was conducted for mouse ISG54. The black scale bar indicates a distance of 500 µm.

Article Snippet: RNA transfection was conducted in a 48-well plate format using the Trans IT-mRNA Transfection kit (Mirus) as per the manufacturer's instructions.

Techniques: Transfection, Construct, Infection, Virus, Control, Comparison, Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Immunohistochemistry, Staining

Journal: The EMBO Journal

Article Title: hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

doi: 10.1038/sj.emboj.7601450

Figure Lengend Snippet: hRpn13 promotes isopeptidase activity of UCH37. (A) UCH37 binds to S5a in vitro. Purified GST or GST-fused UCH37 was incubated with purified recombinant His-tagged S5a for 1 h. Pull-down assays using GSH beads were then performed. The protein levels were analyzed by Western blot. (B) Recombinant hRpn13 stimulates the ubiquitin-amc (Ub-amc) hydrolysis by UCH37. In the presence of 0.5 μM of Ub-amc, 0.1 nM of UCH37 was incubated with 0 or 10 nM of the full-length hRpn13 (FL, from bacteria), the C-terminal half of hRpn13 (Δ1–200, from bacteria), or the full-length hRpn13 expressed in insect cells (FL-ins). Ub-amc hydrolysis (in arbitrary units) was determined during incubation for 30 min by monitoring the release of amc. (C) Unlike hRpn13, pure S5a/Rpn10 cannot promote the isopeptidase activity of UCH37 in vitro. UCH37 at indicated concentrations, was incubated with 0 (circle) or 10 nM of hRpn13 (square, expressed in insect cells), S5a (cross), or hRpn13 plus 10 nM of Ub-aldehyde (triangle). Ub-amc hydrolysis (in arbitrary units) was determined as in (B). (D) hRpn13 does not increase the isopeptidase activity of the 26S proteasome or UCHL3. Ub-amc was incubated (as in 5B) without (control) or with 10 nM of purified hRpn13 in the presence of UCH37 (0.1 nM), the 26S proteasome (0.24 μg/ml, about 0.1 nM), or UCHL3 (0.01 nM). Ub-amc hydrolysis (in arbitrary units) was determined as in (B). (E) hRpn13 decreases the levels of ubiquitin conjugates in cells. Left, 293T cells were transfected with an empty vector or Myc/His6-tagged hRpn13. Right, 293T cells were transfected with pBS/U6/GFP (GFP siRNA) or pBS/U6/hRpn13 (hRpn13 siRNA). The cells were incubated for 2 days after transfection, and the contents of hRpn13, β-actin, and large ubiquitin conjugates (molecular weights >191 kDa) were assayed by Western blot.

Article Snippet: In addition, a pool of four siRNA oligos specific for hRpn13 (Smartpool) and a nontargeting siRNA oligo were purchased (Dharmacon Inc.) and transfected using trans-IT-TKO transfection kit (Mirus Inc.).

Techniques: Activity Assay, In Vitro, Purification, Incubation, Recombinant, Western Blot, Ubiquitin Proteomics, Bacteria, Control, Transfection, Plasmid Preparation

Journal: The EMBO Journal

Article Title: hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

doi: 10.1038/sj.emboj.7601450

Figure Lengend Snippet: hRpn13 content influences rates of degradation of short-lived proteins in 293T cells. (A) Overexpression of hRpn13 or its C-terminal region reduces the degradation of short-lived proteins. 293T cells were transfected with an empty vector, hRpn13 or its C-terminal region (hRpn13-Δ1–200). After 2 days, the rate of degradation of cellular proteins was determined by pulse-chase analysis using [3H]tyrosine. A similar small reduction in degradation was seen in at least three experiments. (B) Decreasing the levels of hRpn13 also slows the degradation of short-lived proteins. 293T cells were transfected with pBS/U6/GFP (GFP siRNA) or pBS/U6/hRpn13 (hRpn13 siRNA). Then, degradation of short-lived proteins was determined as in (A). (C) Overexpression or knockdown of hRpn13 reduces the degradation of the model N-end rule substrate, Ub-R-GFP. Left, 293T cells were cotransfected with Ub-R-GFP, GFP, and hRpn13 (or empty vector). Right, 293T cells were cotransfected with Ub-R-GFP, GFP, and siRNA oligos (100 nM) for hRpn13 (or nontargeting siRNA oligos). The cells were incubated for 3 days after transfection, and the protein levels were analyzed by Western blot. (D) Degradation of the natural proteasomal substrate, ErbB3, is reduced by overexpression of hRpn13 or its C-terminal half. 293T cells were cotransfected with ErbB3, EGFR, and hRpn13 (or hRpn13-Δ1–200 or empty vector), and incubated for 2 days after transfection. Protein levels were analyzed by Western blot.

Article Snippet: In addition, a pool of four siRNA oligos specific for hRpn13 (Smartpool) and a nontargeting siRNA oligo were purchased (Dharmacon Inc.) and transfected using trans-IT-TKO transfection kit (Mirus Inc.).

Techniques: Over Expression, Transfection, Plasmid Preparation, Pulse Chase, Knockdown, Incubation, Western Blot

Journal: Molecular cell

Article Title: Distinct Binding Preferences between Ras and Raf Family Members and the Impact on Oncogenic Ras Signaling

doi: 10.1016/j.molcel.2019.09.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For protein depletion experiments, lentiviral particles expressing the desired targeting constructs were generated by co-transfecting the pLKO.1 or pLentiCRISPRv2 constructs with the MISSION lentiviral packaging mix (Sigma) into 293T cells using the Mirus Trans-IT lenti transfection kit.

Techniques: Recombinant, Transfection, Clone Assay, Western Blot, Live Cell Imaging, CRISPR, Plasmid Preparation, Construct, Software