ren luciferase activity  (BMG Labtech)

Journal: Nucleic Acids Research

Article Title: RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes

doi: 10.1093/nar/gkaa646

Figure Lengend Snippet: ( A ) Schematic representation of eIF4G homologs highlighting functional domains. The numbering is based on Gene Accession NM_182917.4 (eIF4GI), NM_001198802.2 (eIF4GII) and NM_001418.4 (eIF4GIII). Domain organization is based on Marintchev et al. . ( B ) Schematic diagram illustrating internal recruitment of 40S ribosomes and associated initiation factors (represented by a yellow star) via a λN-4G fusion, based on the tethering assay . ( C ) Schematic representation of retroviral-based expression system for ectopic production of eIF4GI in cells. Shown is the vector designed to express the middle domain of eIF4GI (amino acids 653–1131) fused to two λN domains. ( D ) Western blot denoting expression of λ–4GIm in translation extracts prepared from MSCV/λ–4GIm—infected K-2 cells (K-2/λ–4GIm). ( E ). Top: Schematic representation of bicistronic reporters used in this study. Shown are variations in the inter-cistronic region (ICR) tested. BBoxes or scrambled sequence controls (Scr) are present one (1×), three (3×) or six (6×) times, with a spacing of 26 or 52 nucleotides. Bottom: Luciferase production following in vitro translation of 10 μg/ml of the indicated mRNA reporters in either K-2 or K-2/λ-4GIm extracts. Values are normalized to the Scr control mRNAs of the same length and copy number (see ) and represent the average of 5 biological replicates, with each experiment performed in technical duplicates. ± SD. ns, not significant, ** P = 0.02. ( F ). Top: Schematic representation of monocistronic reporters used in this study. Bottom: Luciferase production following in vitro translation of 4 μg/ml of the indicated mRNA reporters in the indicated extracts. Values are normalized to the Scr control mRNAs of the same length. n = 5 biological replicates, ± SD.

Article Snippet: Cells were lysed 12–16 h later using Passive Lysis Buffer (Promega) and FF and Ren luciferase activity determined on a Fluostar 96-well plate reader BMG Labtech ( ).

Techniques: Functional Assay, Retroviral, Expressing, Plasmid Preparation, Western Blot, Infection, Sequencing, Luciferase, In Vitro, Control

Journal: Nucleic Acids Research

Article Title: RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes

doi: 10.1093/nar/gkaa646

Figure Lengend Snippet: ( A ) Schematic diagram illustrating the order of transfection events in the in cellula tethering assay. Cells are first transfected with expression vectors driving synthesis of λ-fusions and GFP. One day later, mRNA reporters are transfected and luciferase values determined 12–16 h later. ( B ) Schematic representation of eIF4E and eIF4G expression constructs. Numbers in parenthesis denote amino acid positions for the eIF4G homologs. Functional domains defined in Figure are shown for reference. ( C ) Luciferase production following co-expression of the indicated monocistronic mRNAs and MSCV expression vectors in HEK293T cells. Values are normalized to cells having received empty MSCV expression vector. n = 3, ± SD. * P < 0.01; **, 0.05> P >0.01. ( D ) Western blot documenting expression levels of λ-fusions in transfected HEK293T cells. ( E ) Assessment of A-capped 3×BBox-FF mRNA levels in transfected cells by RT-qPCR. Values are expressed relative to cells having received empty MSCV/λ-SVgfp expression vector. n = 3, ±SD.

Article Snippet: Cells were lysed 12–16 h later using Passive Lysis Buffer (Promega) and FF and Ren luciferase activity determined on a Fluostar 96-well plate reader BMG Labtech ( ).

Techniques: Transfection, Expressing, Luciferase, Construct, Functional Assay, Plasmid Preparation, Western Blot, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes

doi: 10.1093/nar/gkaa646

Figure Lengend Snippet: ( A ) Luciferase production following co-expression of the indicated MSCV expression vectors in HEK293T cells with 3×BBox mRNA. n = 3, ±SD. ( B ) Western blot documenting expression levels of λN fusions in transfected HEK293T cells. ( C ) Immunoprecipitations performed from cell extracts expressing the indicated FLAG-tagged λN-4E fusion proteins. Following SDS-PAGE, Western blots were performed with antibodies targeting eIF4GI. ( D ) Luciferase production following co-expression of the indicated MSCV-eIF4A expression vectors in HEK293T cells with 3×BBox mRNA. n = 3, ±SD. ( E ) Western blot documenting expression levels of λN fusions in transfected HEK293T cells. ( F ) Western blot documenting subcellular localization (C, cytoplasmic; N, nuclear) of the indicated λN fusions in transfected HEK293T cells. eEF2 and hnRNPA1 are used as loading control for cytoplasmic and nuclear fractionation, respectively.

Article Snippet: Cells were lysed 12–16 h later using Passive Lysis Buffer (Promega) and FF and Ren luciferase activity determined on a Fluostar 96-well plate reader BMG Labtech ( ).

Techniques: Luciferase, Expressing, Western Blot, Transfection, SDS Page, Control, Fractionation

Journal: Nucleic Acids Research

Article Title: RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes

doi: 10.1093/nar/gkaa646

Figure Lengend Snippet: Structure-guided approach to generating orthogonal eIF4G:eIF4A pairs. ( A ) A model of the interface between human eIF4A1:eIF4GI was created by transposing the human sequence onto the yeast (y) structure (PDB accession number 2VSO) . Overview of the yeIF4G:eIF4A complex with human residues modelled at the interface. i . Residue pair eIF4GI(S738):eIF4A1(D265) forms a putative interaction in human. The identity and numbering in yeast homologs of the relevant residues are indicated in parenthesis. The amino acids targeted in human eIF4A1, as well as corresponding mutations made, are indicated. Note that the side chain of yK582 is disordered. ii . eIF4GI residues R764 and S767 form a putative interaction with eIF4A1 residue E268 in human. iii . eIF4GI residues T773 and eIF4A1 residue T298 form a putative interaction in human. iv . eIF4GI residue Q779 and/or eIF4GI residue Q783 forms a putative interaction with eIF4A1 D296 in human. v . eIF4GI residues D982 and eIF4A1 residue R45 form a putative interaction in human. ( B ) Luciferase production following co-expression of the indicated constructs in HEK293T cells with 3xBBox mRNA. Arrows refer to the increase in signal obtained with the obligate dimer pair compared to signal obtained with only the eIF4G mutant—only the largest increase is denoted in this way. Values are normalized to cells having received empty MSCV expression vector. n = 3, ± SD. ( C ). Western blot documenting expression levels of recombinant fusions in transfected HEK293T cells.

Article Snippet: Cells were lysed 12–16 h later using Passive Lysis Buffer (Promega) and FF and Ren luciferase activity determined on a Fluostar 96-well plate reader BMG Labtech ( ).

Techniques: Sequencing, Residue, Luciferase, Expressing, Construct, Mutagenesis, Plasmid Preparation, Western Blot, Recombinant, Transfection