Total RNA from HAP1 cells (isolated as described above) was incubated with 75 or 150 pmol recombinant hENDOV as described above for the activity assays. A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls. Equal volume of formamide loading dye was added and the samples were heated at 50°C for 5 min before separation by 15% denaturating PAGE (7 M urea and 1x taurine) at 200 V for 50 min in 1x taurine. One of the gels was stained with ethidium bromide to check for RNA integrity. The RNA was transferred to a nylon membrane (Hybond XL, GE Healthcare, RPN203S) by electroblotting in 1x taurine at 5 V for 1 h at room temperature. RNA was UV-crosslinked to the membranes (120 mJ/cm-2 in a CL-1000 UV-Crosslinker, UVP). The Northern Max kit (Thermo Fisher Scientific, AM1940) was used for prehybridization, hybridization and washing steps as described by the manufacturer. 32P 5’-labelled oligonucleotides (Eurofins) complementary to the tRNAs: AlaAGC5’, ArgACG5’ and ValAAC5’ were used as probes (sequences available on request). Hybridization signals were analyzed by phosphorimaging and quantified by ImageQuant TL software using background substraction rolling ball. Hybridized probes were removed from the filters by boiling in 0.1% SDS.
Cl 1000 uv crosslinker
Manufactured by Analytik Jena
Sourced in United States, United Kingdom
The CL-1000 UV Crosslinker is a laboratory instrument designed for UV radiation exposure. It provides controlled and reproducible UV irradiation for various applications, such as DNA and RNA crosslinking. The CL-1000 features a built-in UV lamp and a programmable timer to ensure precise exposure times.
Automatically generated - may contain errors
Lab products found in correlation
Hybond xl, by GE Healthcare (3 mentions)
T7 rna polymerase, by Promega (2 mentions)
Northernmax kit, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ultrahyb oligo buffer, by Thermo Fisher Scientific (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Pharos fxtm plus molecular imager, by Bio-Rad (1 mentions)
Hybond nx membrane, by Cytiva (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Welgene (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Fv1000mp, by Olympus (1 mentions)
Phosphorimaging screen, by Fujifilm (1 mentions)
Multi gauge v2, by Fujifilm (1 mentions)
Trans blot electrophoretic transfer cell, by Bio-Rad (1 mentions)
Dna oligo probes, by Merck Group (1 mentions)
Fla5100 reader, by Fujifilm (1 mentions)
γ 32p atp, by Hartmann Analytic (1 mentions)
Quanti blue reagent, by InvivoGen (1 mentions)
Hek blue ifn α β cells, by InvivoGen (1 mentions)
25 protocols using cl 1000 uv crosslinker
1
Enzymatic analysis of tRNA cleavage by hENDOV
2
UV Cross-linking of PRMT6 with AdoMet
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UV cross-linking of S-adenosyl-L-[methyl-3H] methionine to PRMT6 was performed as previously described [44 (link)]. A CL-1000 UV cross-linker was used (UVP, Upland, CA, U.S.A.). GST-PRMT6 (10 µg) without any competitor or with 200 µM sinefungin, 200 µM licochalcone A, 200 µM AMI-5, respectively, was exposed to UV light (254 nm) at a distance of 1 cm for 30 min at 4°C in the presence of 3.2 µM [3H]AdoMet and 5 mM dithiothreitol in a total volume of 50 µl of PBS. After UV cross-linking, samples were run on SDS–PAGE and subjected to fluorography. Ponceau S staining of the same membrane served a loading control.
3
Northern Blot Analysis of Viral RNAs
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Total RNA was extracted using the TRIreagent (Sigma), as previously described [37 (link)]. Ten micrograms of total RNA were separated on a denaturing 12% polyacrylamide gel (8M urea, 1 × MOPS buffer) and transferred to a Hybond NX membrane (Amersham Biosciences, Piscataway, NJ, USA) with semidry electroblotting [38 (link)]. RNA was crosslinked (1200 × 100 μJ/cm2, 254 nm) to the membrane using a CL-1000 UV Crosslinker (UVP, CA, USA). The membrane was hybridized in an ULTRAhyb-Oligo buffer (Thermo Fisher, Waltham, MA, USA) with γ-32P-ATP labelled hybridization probes detecting 5′ and 3′ ends of the HAdV-4 VA RNAI, HAdV-5 VA RNAI, or tRNA-Lysine (Supplementary Table S2 ). After overnight hybridization, the membrane was washed 3× for 10 min at 42 °C in a 3× SSC, 0.5% SDS buffer followed by a single wash with a 1× SSC, 0.5% SDS buffer for 15 min at 42 °C. Radioactive signals were detected using a Pharos FXTM Plus Molecular Imager and analysed using a Quantity One 4.6.9 (Bio-Rad).
4
Photocrosslinkable MSC-secretome Hydrogel
5
In Vitro Binding and Purification of Cry1-3'UTR-Interacting Proteins
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For in vitro binding assays, [32P]UTP-labelled RNA was transcribed from XbaI-linearized recombinant pSK′ vectors using T7 RNA polymerase (Promega). In vitro binding and UV-crosslinking were performed as previously described (3 (link),27 (link)). Briefly, equal amounts of labelled RNAs were incubated with 15 μg nuclear extracts or 30 μg cytoplasmic extracts of NIH 3T3 cells for 20 min. After incubation, the samples were UV irradiated on ice for 10 min using a CL-1000 UV crosslinker (UVP). Unbound RNA was digested with 5 μl of an RNase cocktail containing RNase A and RNase T1. The reaction mixtures were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.
Streptavidin-biotin RNA affinity purification of Cry1-3′UTR-binding proteins was performed as previously reported (28 (link)). Briefly, cytoplasmic extracts prepared from NIH 3T3 cells were incubated with biotinylated Cry1-3′UTR, and were subjected to streptavidin resin adsorption. Resin-bound proteins were analysed by SDS-PAGE.
Streptavidin-biotin RNA affinity purification of Cry1-3′UTR-binding proteins was performed as previously reported (28 (link)). Briefly, cytoplasmic extracts prepared from NIH 3T3 cells were incubated with biotinylated Cry1-3′UTR, and were subjected to streptavidin resin adsorption. Resin-bound proteins were analysed by SDS-PAGE.
6
Purification and Inactivation of Japanese Encephalitis Virus
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The GI strains of SH7 (GenBank No. MH753129) and SD12 (GenBank No. MH753127), and GIII strains of SH15 (GenBank No. MH753130) and SH19 (GenBank No. MH753131) used in this study were previously isolated from aborted pigs or mosquitoes during 2015–2016[29 (link)]. All JEV strains were plaque-purified three times in BHK cells and subsequently amplified in BHK cells at 0.1 MOI. The supernatants collected from the infected BHK cells were subjected to sucrose gradient ultracentrifugation, as described previously[62 (link)]. The white matter of fraction containing JEV was collected and diluted with phosphate buffered saline (PBS) in an ultracentrifuge tube. After centrifugation at 200,000 x g at 4°C for 1.5 h, the pellet was resuspended in PBS and dissolved overnight at 4°C. The virus stocks were stored at −80°C until use and the viral titer was measured in BHK cells. To inactivate JEV, virus was irradiated using a CL-1000 UV crosslinker (UVP, California, USA) at 500 mJ/cm2 for 15 min. The inactivation was confirmed by inoculation of the irradiated virus into BHK cells.
7
In-vitro Skin Aging Model
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For developing experimental models of skin aging, Hs68 cells were seeded in 24-well plates and were incubated in a culture medium without FBS for 24 h. Then, the cells were washed with phosphate buffer saline (PBS; WelGENE; Daegu, Korea), stimulated with 20 mJ/cm2 UVB using a CL-1000 UV crosslinker (UVP; Cambridge, UK) or 1 μM dexamethasone (Sigma-Aldrich; Seoul, Korea), and further incubated in the same medium for another 24 h.
8
Photochemical Activation of Peptides
9
Northern blotting for RNA detection
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Northern blotting was performed as described in Kruszka et al. (66 (link)). Briefly: 30 µg of RNA (per sample) isolated from transfected tobacco leaves was loaded on 8 M denaturing urea polyacrylamide gel (15%) in TBE buffer (0.089 M Tris, 0.089 M boric acid, and 0.002 M EDTA, pH 8.0). RNA was then transferred onto the Amersham Hybond-NX nitrocellulose membrane (GE Healthcare) using a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) and fixed using CL-1000 UV Crosslinker (UVP). Prehybridization and hybridization were performed in hybridization buffer (3.5% SDS, 0.375 M sodium phosphate dibasic, 0.125 M sodium phosphate monobasic) at 42 °C with DNA oligo probes (Sigma) labeled at their 5′ ends with γ32P ATP (Hartmann Analytic). U6 was used as a loading control. After washing, the blots were exposed for up to 3 d to a phosphorimaging screen (Fujifilm) and the results were visualized with the Fujifilm FLA5100 reader (Fujifilm) and quantified using Multi Gauge V2.2 (Fujifilm).
10
Extragenic Mutation Screening of NstA Toxicity
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