HEK 293T cells were grown in 6 cm dishes and treated with 15 μM lovastatin (M2147-25MG, Sigma) for about 2 h. Fresh medium containing 400 μCi of mevalonolactone RS-[5-3H] (ART 0315A, ARC) and 15 μM lovastatin was added. Cells were then transiently transfected with FLAG-tagged FBXL2(C417S/C419S) and FBXL2(C420S). After overnight incubation, cells were harvested and lysed with Nonidet P-40 substitute (Roche)-based lysis buffer containing protease inhibitor cocktail (Roche) and sonicated (Sonic Dismembrator, Fisher Scientific). A portion of the lysate was taken as input and the rest was clarified using Protein G Agarose (Invitrogen) followed by overnight immunoprecipitation with anti-flag M2 affinity gel (A2220-Sigma). After washing the M2 gel, elution was carried out with 2X Bolt LDS sample buffer (novex). The samples were run on Bolt 4–12% Bis-Tris Plus gel (Invitrogen) and transferred onto PVDF membrane (Millipore). The membrane was placed under a phosphor screen (28-9564-82, Cytiva) for two weeks and the screen was exposed in a phosphor imager (Amersham Typhoon, GE) for [3H] detection. The PVDF membrane was finally immunoblotted for FLAG-tagged FBXL2(C417S/C419S) and FBXL2 (C420S) expression using rabbit anti-FLAG antibody (F7425-Sigma).
Amersham typhoon
Manufactured by GE Healthcare
Sourced in Sweden, United States
The Amersham Typhoon is a phosphor imager designed for high-resolution imaging of radiolabeled samples. It utilizes a photomultiplier tube and a laser to detect and quantify radioactive signals from samples, such as DNA, RNA, and protein gels, blots, and arrays. The Amersham Typhoon provides high-sensitivity and high-resolution digital images of radioactive samples.
Automatically generated - may contain errors
Lab products found in correlation
F7425, by Merck Group (4 mentions)
Anti flag m2 affinity gel, by Merck Group (3 mentions)
Imagequant tl, by GE Healthcare (3 mentions)
Hybond, by Cytiva (3 mentions)
Storage phosphor screen, by GE Healthcare (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protein g agarose, by Thermo Fisher Scientific (2 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Bolt lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Sonic dismembrator, by Thermo Fisher Scientific (2 mentions)
M2147 25mg, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Nonidet p 40 substitute, by Roche (2 mentions)
Hybond n1 membrane, by Cytiva (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Perfecthyb plus hybridization buffer, by Merck Group (2 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Fla 5100, by Fujifilm (1 mentions)
41 protocols using amersham typhoon
1
Tracking Mevalonate Metabolism in FBXL2 Mutants
2
GTPase Activity Assay of hGBP3 Variants
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GTPase activity assays of WT hGBP3, truncated, mutant variants were performed using reaction buffer having tris (Sigma) 50 mM (pH 7.5), MgCl2 (Merck) 5 mM, KCl (Merck) 100 mM, DTT (Sigma) 0.2 mM, and a minute quantity of [α-32P] radiolabeled GTP (PerkinElmer). The enzyme and unlabeled GTP concentrations were varied depending on the experiment. The reaction was performed for 30 min at 37 °C and stopped by the addition of 125 mM EDTA (the final concentration). The reaction mixture was then subjected to thin-layer chromatography to separate the products. Polyethyleneimine cellulose sheets with 0.75 M KH2PO4, pH 3.5 buffer were used for this purpose. After separation, the polyethyleneimine cellulose sheets were exposed to a phosphor imaging plate for 12 to 16 h and then quantified using phosphorimagers (FLA-5100, Fujifilm and Amersham Typhoon, GE). The intensities and contrasts of the blots have been adjusted to generate presentable images.
3
EMSA Analysis of TtCmr-RNA Interactions
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EMSAs were performed by incubating 62.5 nM TtCmr complex with 13.3 nM 5′ Cy5-labeled target RNAs (Supplementary Table S1 ) in Cmr binding buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1 mM DTT, 1 mM EDTA). All reactions were incubated for 20 min at 65 °C before electrophoresis on a native 5% (w/v) polyacrylamide gel (PAGE), running at 15 mA. The image was visualized via fluorescent gel scanning (GE Amersham Typhoon).
4
In Vitro Cleavage Assay for TtCmr
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RNA substrates (listed in Supplementary Table S1 ) were either 5′ labeled by T4 polynucleotide kinase (NEB) and 5′ 32P-γ -ATP, after which they were purified from a denaturing PAGE using RNA gel elution buffer (0.5 M Sodium acetate, 10 mM MgCl2, 1 mM EDTA and 0.1% SDS) or ordered with a 5′ Cy5 fluorescent label. In vitro cleavage activity assays were conducted in TtCmr activity assay buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM DTT, 1 mM ATP, and 2 mM MgCl2) using the RNA substrate and 62.5 nM TtCmr. Unless stated otherwise, the reaction was incubated at 65 °C for 1 h. RNA loading dye (containing 95% formamide, dyes left out in case of Cy5 substrates) was added to the samples after incubation and boiled at 95° for 5 min. The samples were run on a 20% denaturing polyacrylamide gel (containing 7 M urea) for about 1–4 h at 15 mA or overnight at a constant of 4 mA. The image was visualized using phosphorimaging or fluorescent gel scanning (GE Amersham Typhoon).
5
PKR Autophosphorylation Assay
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PKR (100 nM) was incubated with specified concentrations of RNA for 10 min at 30°C in 20 mM HEPES (pH 7.5), 100 mM NaCl, 4 mM MgCl2, 1.5 mM DTT, 0.1 mM ATP and 1.5 µCi [ɣ-32P]ATP (PerkinElmer). Reactions were quenched by addition of SDS-sample buffer (188 mM Tris-Cl pH 6.8, 3% SDS, 30% glycerol, 0.01% bromophenol blue, 15% BME) and resolved on 4%–15% PAGE (BioRad). After electrophoresis, gels were fixed in 7% acetic acid for 15 min and then dried. Gels were exposed to a PhosphorImage screen and quantified on a Typhoon Trio PhosphorImager or an Amersham Typhoon (GE Healthcare). Values were normalized to autophosphorylation reactions performed with perfectly duplexed RNA of 106 bp at 10 nM. Autophosphorylation assays were repeated at least three times for each RNA.
6
Kinetic Assay of UBE2N E2-Ubiquitin Conjugation
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20 μM UBE2N was loaded with 20 μM fluorescent UB K63R
(UB*) in the presence of 0.3 μM UBA1 in a buffer containing 50 mM Hepes,
pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 1.5 mM ATP and 0.05 mg/ml BSA.
Loading reactions were incubated for 0.5 h and quenched by adding EDTA to a
final concentration of 30 mM. The reaction was then initiated by adding
UBE2N~UB* (0.5 μM final) to a substrate mix containing 0.5 μM
UBE2V1, 0.5 μM RNF4 RING domain and 35mM amino acid acceptors
(NƐ-acetyl-L-Lysine, L-Serine, L-Dap,
Nɑ-acetyl-L-Ornithine, L-Lysine, D-Lysine,
Nɑ-acetyl-L-Lysine, or L-Homolysine). Reactions were
quenched with either non-reducing or reducing SDS-PAGE sample buffer after 0, 5,
10, 20, 30, 45, 60, 120 or 180 min, and substrates and products were separated
by SDS-PAGE. Gels were scanned on an Amersham Typhoon (GE Healthcare) and the
intensities of all fluorescent bands were quantified using ImageQuantTL (GE
Healthcare). The E2~UB* band intensities were divided by the total fluorescent
intensity in each lane and normalized to the 0 time point. Data were plotted in
GraphPad Prism 8 (GraphPad Software) and fitted to an exponential decay function
using non-linear regression. All reactions were performed in duplicate.Source Data Fig. 1 and
Source Data Extended Data
Fig. 1 contains all gels obtained from this experiment.
(UB*) in the presence of 0.3 μM UBA1 in a buffer containing 50 mM Hepes,
pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 1.5 mM ATP and 0.05 mg/ml BSA.
Loading reactions were incubated for 0.5 h and quenched by adding EDTA to a
final concentration of 30 mM. The reaction was then initiated by adding
UBE2N~UB* (0.5 μM final) to a substrate mix containing 0.5 μM
UBE2V1, 0.5 μM RNF4 RING domain and 35mM amino acid acceptors
(NƐ-acetyl-L-Lysine, L-Serine, L-Dap,
Nɑ-acetyl-L-Ornithine, L-Lysine, D-Lysine,
Nɑ-acetyl-L-Lysine, or L-Homolysine). Reactions were
quenched with either non-reducing or reducing SDS-PAGE sample buffer after 0, 5,
10, 20, 30, 45, 60, 120 or 180 min, and substrates and products were separated
by SDS-PAGE. Gels were scanned on an Amersham Typhoon (GE Healthcare) and the
intensities of all fluorescent bands were quantified using ImageQuantTL (GE
Healthcare). The E2~UB* band intensities were divided by the total fluorescent
intensity in each lane and normalized to the 0 time point. Data were plotted in
GraphPad Prism 8 (GraphPad Software) and fitted to an exponential decay function
using non-linear regression. All reactions were performed in duplicate.
Fig. 1
7
Kinetic Assay of UBE2N E2-Ubiquitin Conjugation
8
Gel Electrophoresis Imaging Protocol
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The samples were run on a 4–15% gel (Bio-Rad). The gel was fixed in 10% acetic acid, 50% methanol for 1 h at room temperature. The gel was rinsed 2× with deionized water. The gel was incubated in deionized water for 30 min at room temperature 2×. The gel was dried at 70°C overnight. The gel was exposed to the phosphor screen and imaged using a GE Amersham Typhoon.
9
In Vivo Cross-linking and Covalent Capture
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Cross-linking was performed as described in (Soh et al., 2019 (link)). Cultures were grown in SMG to OD600 of 0.03–0.04 at 37°C. Cells were mixed with ice and harvested by centrifugation. Sample handling and preparation were performed on ice and cold at every step. Cells were washed in PBSG (PBS with 0.1% [v/v] glycerol). Samples were resuspended in 1 mL PBSG. 1.25 OD600 equivalents were taken and pelleted by centrifugation. Pellets were resuspended in 30 μL PBSG. Cross-linking agent (SMCC (Thermo) or BMOE (Thermo) was added to a final concentration of 0.5 mM and mixed by vortexing. Reactions were incubated on ice for 10 minutes and then quenched by the addition of 0.5 mM final concentration 2-mercaptoethanol with subsequent incubation for 2 minutes. Samples were supplemented with additives at the indicated final concentrations: benzonase (750 U/mL; Sigma), 5 μM HaloTag-TMR ligand (Promega), Ready-Lyse Lysozyme (47 U/μL; Epicentre), and 1× PIC (Sigma-Aldrich). Samples were incubated at 37°C for 30 minutes under light protection. Samples were supplemented with LPS loading dye and denatured at 70°C for 5 minutes. Samples were run on 3-8% Tris-acetate gels (Invitrogen) at a constant power output of 35 mA at 4°C. In-gel fluorescence was imaged using an Amersham Typhoon (GE Healthcare) with a Cy3 DIGE filter. Quantification was performed using ImageQuant (GE Healthcare).
10
Asymmetric Nucleosome Remodeling Assay
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We prepared hexasomes according to published methods (30 (link),31 (link)). Hexamers were reconstituted, containing either H2A/H2B dimers or biotin-modified H2A/H2B S112C dimers, then converted to full nucleosomes with modified or native dimers, respectively, to generate asymmetrically modified nucleosomes. Converted nucleosomes were purified by sucrose gradients. Remodeling reactions were carried out as described above, in the presence or absence of streptavidin, and reactions terminated by the addition of 200 ng plasmid DNA. Reactions were digested with 10 u HindIII for 60 min at 37°C then loaded directly onto 5% acrylamide nucleoprotein gels and the wet gels imaged by fluorography (GE Amersham Typhoon). The amount of products before and after HindIII digestion was quantified by densitometric analysis of the fluorographs and the fraction remaining uncut by HindIII calculated and plotted.
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