Journal: eLife

Article Title: Mutations in L-type amino acid transporter-2 support SLC7A8 as a novel gene involved in age-related hearing loss

doi: 10.7554/eLife.31511

Figure Lengend Snippet: Hearing phenotype of C57BL6/J-129Sv Slc7a8 knockout mice. ( A ) Representative image of western bloting of total membranes from kidney, brain and intestine of wild-type (+/+) and Slc7a8 knock out (-/-) mice in the absence (-) or presence (+) of 100 mM dithiothreitol reducing agent (DTT) of three independent biological samples for both sexes (male and female). Protein (50 μg) were loaded in 7% acrylamide SDS-PAGE gel. Molecular mass standard (KDa) are indicated. Red arrows point SLC7A8/CD98hc heterodimer band as well as the light subunit SLC7A8. Upper panel: Rabbit anti-SLC7A8. Bottom panel: Mouse anti-βactin. ( B ) Pre-Pulse Inhibition of the acoustic startle response (PPI). Mean and SEM are represented. Pulse: 120 dB single pulse. Pre-pulse inhibition test: six different pre-pulse intensities (70 to 90 dB) in pseudo random order with 15 s inter-trial intervals. Wild type (white circles, n = 19) and Slc7a8 −/− (blue circles, n = 15) from 4- to 7-month-old are represented. Significant differences were determined using paired Student’s t-test, ***p

Article Snippet: For specific SLC7A8 light subunit detection, samples were run in the presence of 100 mM of dithiothreitol (SigmaAldrich Ref:D9779).

Techniques: Knock-Out, Mouse Assay, Western Blot, SDS Page, Inhibition

Journal: Journal of Virology

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis

doi: 10.1128/JVI.76.20.10417-10426.2002

Figure Lengend Snippet: G13 and U14 in the NS1 5′ UTR are required for GRSF-1 binding. Wild-type (Wt) GST-GRSF-1 (1.25 × 10 −11 mol) was incubated with 10 6 dpm of radiolabeled wild-type NS1 5′ UTR RNA in the absence or presence of increasing concentrations (10- or 100-fold molar excess) of unlabeled wild-type (lanes 2 to 4), U14C (lanes 5 to 7), or G13C/U14C (lanes 8 to 10) competitor RNAs in 5 mM HEPES (pH 7.6)-25 mM KCl-2 mM MgCl 2 -5% glycerol-100 mM NaCl-2 mM dithiothreitol-20 U of RNasin (Promega) at 30°C for 15 min. Samples were then exposed to UV light (1.0 J) in a Stratalinker (Stratagene), followed by incubation with 10 mg of RNase A per ml and 2 U of RNase T 1 at 37°C for 30 min. Samples were suspended in 1× Laemmli buffer and separated by SDS-10% PAGE, and the gel was dried and visualized with a Storm 850 phosphorimager (Molecular Dynamics). These data are representative of three independent experiments.

Article Snippet: The S10 lysate was supplemented with 0.0156 mg of tRNA per ml, 0.62 mM ATP, 0.037 mM GTP, 6.22 mM creatine phosphate, 0.0156 mg of creatine kinase per ml, 11.8 mM HEPES (pH 7.6), 1.24 mM dithiothreitol, 15.6 μM complete amino acid mixture (Promega), and 0.156 mM spermidine.

Techniques: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis

Journal: Journal of Virology

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis

doi: 10.1128/JVI.76.20.10417-10426.2002

Figure Lengend Snippet: GRSF-1 interacts with specific sequences within the influenza virus mRNA 5′ UTR as detected by gel mobility shift analysis. Recombinant GRSF-1 (100 ng) was incubated with radiolabeled NP-c, NPD3, NPD6, NPD9, NP12, NP14, M1, NA, PB1, and NP-B RNAs (10 5 dpm) in buffer that contained 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl 2 , 3.8% glycerol, 100 mM NaCl, 0.02 mM dithiothreitol, 2 mM GTP, and 1.5 mM ATP at 30°C for 20 min. The resulting RNA-protein complexes were resolved by 5% native PAGE and visualized by autoradiography. These data are representative of two independent experiments.

Article Snippet: The S10 lysate was supplemented with 0.0156 mg of tRNA per ml, 0.62 mM ATP, 0.037 mM GTP, 6.22 mM creatine phosphate, 0.0156 mg of creatine kinase per ml, 11.8 mM HEPES (pH 7.6), 1.24 mM dithiothreitol, 15.6 μM complete amino acid mixture (Promega), and 0.156 mM spermidine.

Techniques: Mobility Shift, Recombinant, Incubation, Clear Native PAGE, Autoradiography

Journal: Journal of Virology

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis

doi: 10.1128/JVI.76.20.10417-10426.2002

Figure Lengend Snippet: Second RRM of GRSF-1 is required for mRNA binding in vitro. (A) Coomassie blue-stained denaturing 12% polyacrylamide gel of equal moles (1.25 × 10 −11 mol) of wild-type and mutant GST-GRSF-1 fusion proteins purified by glutathione affinity chromatography from E. coli . (B) UV cross-linking analysis of thrombin-cleaved GRSF-1 (rGRSF-1) and intact wild-type (Wt) and mutant GST-GRSF-1 fusion proteins (1.25 × 10 −11 mol) incubated with radiolabeled NP 5′ UTR RNA (10 6 dpm) in 5 mM HEPES (pH 7.6)-25 mM KCl-2 mM MgCl 2 -5% glycerol-100 mM NaCl-2 mM dithiothreitol-20 U of RNasin (Promega) at 30°C for 15 min. Samples were then exposed to UV light (1.0 J) in a Stratalinker (Stratagene), followed by incubation with 10 mg of RNase A per ml and 2 U of RNase T 1 at 37°C for 30 min. Samples were suspended in 1× Laemmli buffer and separated by SDS-10% PAGE, and the gel was dried and visualized with a Storm 850 phosphorimager (Molecular Dynamics). These data are representative of two independent experiments.

Article Snippet: The S10 lysate was supplemented with 0.0156 mg of tRNA per ml, 0.62 mM ATP, 0.037 mM GTP, 6.22 mM creatine phosphate, 0.0156 mg of creatine kinase per ml, 11.8 mM HEPES (pH 7.6), 1.24 mM dithiothreitol, 15.6 μM complete amino acid mixture (Promega), and 0.156 mM spermidine.

Techniques: Binding Assay, In Vitro, Staining, Mutagenesis, Purification, Affinity Chromatography, Incubation, Polyacrylamide Gel Electrophoresis

Journal: Journal of Virology

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis

doi: 10.1128/JVI.76.20.10417-10426.2002

Figure Lengend Snippet: GRSF-1 binds the conserved A-box of the influenza virus NP 5′ UTR. Shown here is a representative autoradiogram of RNA gel shift analysis of radiolabeled influenza virus NP (lanes 1 to 7) or NP-A (lanes 8 to 14) 5′ UTR RNA incubated with increasing concentrations (50, 100, and 200 ng) of thrombin-cleaved (rGRSF-1) or uncleaved GST-GRSF-1. Proteins were incubated with radiolabeled RNA (100,000 dpm) in buffer that contained 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl 2 , 3.8% glycerol, 100 mM NaCl, 0.02 mM dithiothreitol, 2 mM GTP, and 1.5 mM ATP at 30°C for 20 min. Samples were then electrophoresed on a 5% polyacrylamide gel at 4°C and visualized by autoradiography. These data are representative of two independent experiments.

Article Snippet: The S10 lysate was supplemented with 0.0156 mg of tRNA per ml, 0.62 mM ATP, 0.037 mM GTP, 6.22 mM creatine phosphate, 0.0156 mg of creatine kinase per ml, 11.8 mM HEPES (pH 7.6), 1.24 mM dithiothreitol, 15.6 μM complete amino acid mixture (Promega), and 0.156 mM spermidine.

Techniques: Electrophoretic Mobility Shift Assay, Incubation, Autoradiography