trex tm system  (Addgene inc)

Journal: Nature Communications

Article Title: Exon-junction complex association with stalled ribosomes and slow translation-independent disassembly

doi: 10.1038/s41467-024-48371-5

Figure Lengend Snippet: a The 3D structure of an in vitro assembled EJC PDB2J0S shows that MAGOH N-terminus is close to Y14 C-terminus and that Magoh C-terminus is close to eIF4A3 C-terminus; b MAGOH stably binds Y14, SmBiT fused to C-terminus (C) of Y14 interacts with LgBiT fused to N-terminus (N) of MAGOH thereby recapitulating NanoBiT luciferase activity; c MAGOH and eIF4A3 interact only when belonging to an assembled EJC, luciferase activity is recapitulated if LgBiT and SmBiT are fused to their respective C-termini (C); d , e relative luciferase activities (RLUs) obtained following transient transfection in HEK 293 cells; f , g HEK293 derived cell lines stably expressing eIF4A3-SmBiT (OB2 cells) or MAGOH-LgBiT (OB3 cells) are transiently transfected with plasmids expressing either wild-type or mutant HA-MAGOH-LgBiT or Flag-eIF4A3-SmBiT; data for d – g are from at least two biological replicates. Western blots probe the expression of tagged transfected proteins, GAPDH is a control for equal amount of cells loaded in every lane. GAPDH, eIF4A3, MAGOH and Y14 proteins are detected on Western blots with specific antibodies diluted 1/1000. Source data are provided in the Source Data file.

Article Snippet: HEK293 Trex TM (Invitrogen #R) cells are transfected by JET Prime.

Techniques: In Vitro, Stable Transfection, Luciferase, Activity Assay, Transfection, Derivative Assay, Expressing, Mutagenesis, Western Blot

Journal: Nature Communications

Article Title: Exon-junction complex association with stalled ribosomes and slow translation-independent disassembly

doi: 10.1038/s41467-024-48371-5

Figure Lengend Snippet: a Lysates from cells coexpressing MAGOH-LgBiT and eIF4A3-SmBiT (OB9 cells) are fractionated on sucrose gradients. Fractions (48 × 0.25 ml) from HEK 293 EJC-NanoBiT expressing cells were collected after a sucrose gradient (black curve - mean of 3 biological replicates normalized by their total luciferase activity). Mean values +/- standard deviation are plot. b Fractions from genuine HEK 293 cell lysates (12 × 1 ml fractions) were collected and analysed by Western blot. Fractions 5 to 9 in 2b correspond to fractions 16 to 36 in 2a, 2d and 2e. eIF4A3, Y14 and MAGOH are detected by Western blot in input fractions and in eIF4A3 immunoprecipitates. c Fragment Analyzer profiles of RNAs isolated from precipitates with (red curve) or without (black curve) anti-HA antibodies from pooled 3–12 fractions (12 fractions) of ultracentrifuged HA-eIF4A3 cell lysates. eIF4A3, MAGOH and Y14 proteins are detected on Western blots with specific antibodies diluted 1/1000. d Ultracentrifugation profiles of EJC-NanoBiT expressing cells treated (pink) or not (black) with cycloheximide (100 μg/ml) for 60 min. e Ultracentrifugation profiles of EJC-NanoBiT expressing cells treated (green) or not (black) with harringtonine (2 μg/ml) for 60 min. All lysates are fractionated on 11–54% sucrose gradients. RNA in 2b is detected by SYBR green fluorescence. RNA content in 2a, 2d and 2e in 48 × 0.25 ml fractions, is monitored by optical densities at 254 nm (dotted curves) while NanoBiT luciferase activities are shown as continuous lines. Positions of 40 S, 60 S, 80 S disomes (D) and trisomes (T) are indicated. f The increased EJC particle size upon treatment with translation inhibitors is attributed to the additional loading of ribosomes arrested at weak non canonical initiation codon or frozen just after initiation and preribosomes queuing upstream. Source data are provided in the Source Data file.

Article Snippet: HEK293 Trex TM (Invitrogen #R) cells are transfected by JET Prime.

Techniques: Expressing, Luciferase, Activity Assay, Standard Deviation, Western Blot, Isolation, SYBR Green Assay, Fluorescence

Journal: Nature Communications

Article Title: Exon-junction complex association with stalled ribosomes and slow translation-independent disassembly

doi: 10.1038/s41467-024-48371-5

Figure Lengend Snippet: a , b Lysates from cells coexpressing MAGOH-LgBiT and eIF4A3-SmBiT (OB9 cells) are fractionated on sucrose gradients. Luciferase activity (RLUs) and SYBR green fluorescence at 540 nm are measured in gradient fractions. a Control lysates (black), lysates with NaCl (600 mM) (light blue), b RNase (purple) or both RNAse and NaCl (orange) are added to lysates from EJC-NanoBiT expressing cells. c Luciferase activity in crude lysates supplemented with 600 mM NaCl (blue) or NaCl and RNase (orange). RNA in 3a, 3b and 3c are detected by SYBR green fluorescence emission at 540 nm (dots) and NanoBiT luciferase activities (continuous lines) are measured in 48 × 0.25 ml gradient fractions. d Lysates from HEK293 cells are fractionated on 11-54 % sucrose gradients. EJC core proteins are detected by Western blot in input fractions (top) or co-immunoprecipitated from the same fractions by anti-eIF4A3 (below). eIF4A3, MAGOH and Y14 proteins are detected on Western blots with specific antibodies diluted 1/1000. All these experiments have been repeated twice with similar results. Source data are provided in the Source Data file.

Article Snippet: HEK293 Trex TM (Invitrogen #R) cells are transfected by JET Prime.

Techniques: Luciferase, Activity Assay, SYBR Green Assay, Fluorescence, Expressing, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: The low affinity neurotrophin receptor CD271 regulates phenotype switching in melanoma

doi: 10.1038/s41467-017-01573-6

Figure Lengend Snippet: CD271 transient overexpression in vitro reveals a reversible phenotype. a Schematic representation of the inducible vector (TetON) to overexpress CD271 in melanoma cell lines. The viral backbone carries the green fluorescent protein (GFP) and infrared fluorescent protein (iRFP) reporters under the SV40 and PGK promoters respectively, as well as the CD271 overexpression cassette (CMVTOCD271) or an empty vector (CMVTOEV) under the inducible CMVTetOperon (CMVTO) promoter. The overexpression of the gene is controlled by doxycycline and is based on the TRex TM system (pLenti CMV TetR Blast, Addgene no. 17492). b Crystal violet staining of adherent cells (upper panels) and suspension cells forced to reattach on fibronectin-coated plates (lower panels). Not treated (day 0), treated with doxycycline for 24 h (day 1), and subsequently released from doxycycline for 48 h (day 4). c Quantification of cell numbers of b ( n = 3, P values > 0.05, ≤ 0.01). Error bars indicate S.D. All experiments done with cell line M010817

Article Snippet: The overexpression of the gene is controlled by doxycycline and is based on the TRex TM system (pLenti CMV TetR Blast, Addgene no. 17492). b Crystal violet staining of adherent cells (upper panels) and suspension cells forced to reattach on fibronectin-coated plates (lower panels).

Techniques: Over Expression, In Vitro, Plasmid Preparation, Staining, Suspension