Amersham typhoon biomolecular imager

Binding reactions contained 50 nM of labeled RNA and increasing concentrations of RNF219-C in a total reaction volume of 15 µl of binding buffer (20 mM PIPES pH 6.8, 40 mM NaCl, 10 mM KCl, 2 mM Mg(OAc)₂, 3% [w/v] Ficoll 400, 0.05% [v/v] NP-40, 0.03% [w/v] Orange G). The proteins were incubated for 20 min with RNA at 37 °C. The RNA-protein complexes were analyzed by electrophoresis on two gel types: 6% nondenaturing polyacrylamide gel at 10 V/cm and 2% TBE/agarose gel at 10 V/cm. Gels were imaged using an Amersham Typhoon Biomolecular Imager (Cytiva).

Samples of extracellular proteins (23 μg) were separated on 10% NuPAGE gels (Invitrogen, USA) by lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis (PAGE). Upon electrophoresis, the separated proteins were stained with SimplyBlue SafeStain (Life Technologies, CA).
For Western blotting analyses of LtxA production, the separated proteins were transferred from the gel to a Protran nitrocellulose transfer membrane (Whatman, Germany) by semidry blotting. Subsequently, the membrane was incubated with the LtxA-specific monoclonal antibody 83 (30 (link)). LtxA-specific protein bands were visualized with an IRDye 800CW-labeled secondary goat-anti mouse antibody and scanning of the membrane with an Amersham Typhoon biomolecular imager (Cytiva, Medemblik, the Netherlands).

Southern blotting was essentially performed according to our previously reported methods (85 (link)). Briefly, lower molecular weight (Hirt) DNA samples were prepared from cells as described (75 (link)), and resolved in 1% agarose gel. The separated DNAs were transferred onto a nitrocellulose membrane (#1212590; GVS North America) and probed with an [α-³²P]-dCTP-labeled probe of the AAV2 repcap gene. In some experiments, the blot was additionally probed for mitochondrial DNA (mtDNA) using a specific probe (32 (link)). After overnight exposure with a storage phosphor screen, hybridization signals were captured and visualized on an Amersham Typhoon Biomolecular Imager (Cytiva), and quantified using ImageQuant Tl (IQTL) 8.2 (Cytiva).

Telomerase primer extension assays were carried out as previously described^{9 (link),36 (link)}. Telomerase sample was incubated in 20-μL reactions containing 50 mM Tris-acetate pH 8.0, 4 mM MgCl₂, 5 mM DTT, 250 μM dTTP, 250 μM dATP, 5 μM unlabeled dGTP, 0.1 μM α-³²P-labeled dGTP (3,000 Ci mmol⁻¹, 10 mCi ml⁻¹) (Hartmann Analytic, Cat# FP-204) and 500 nM DNA reaction primer (T₂AG₃)₅. The reactions were performed at 30 °C for 40 min and stopped with 50 mM Tris HCl pH 7.5, 20 mM EDTA, and 0.2% SDS. DNA was extracted with phenol:chloroform:isoamyl alcohol (ThermoFisher, Cat# 17909), followed by ethanol precipitation with a ³²P-labelled 18 nucleotide (nt) oligonucleotide as a recovery control (RC). Samples were resolved on a 10.5% denaturing polyacrylamide TBE gel. The gel was dried at 80 °C for 60 min, exposed on a phosphorimager screen, and imaged using an Amersham Typhoon Biomolecular Imager (Cytiva). The telomerase activity assay was repeated three times independently. Quantification analysis was performed using ImageQuant (Cytiva), Microsoft Excel, and Prism GraphPad. The activity of the mutants was calculated as the ratio of the RC-normalized counts (total counts over the counts of the RC) and the RC-normalized counts of the wild-type. The standard error of the mean (SEM) and the pairwise one-tailed t-test was calculated in Microsoft Excel.

A total of 400 nM Pol γ and 400 nM duplex DNA were mixed on ice in reaction buffer [25 mM Hepes (pH 7.5), 140 mM KCl, 5% glycerol, 1 mM EDTA (pH 8.0), and BSA (100 μg/ml)] and prewarmed at 37°C for 5 min. Reactions were initiated by adding an equal volume of reaction buffer containing 20 mM MgCl₂ and 100 μM dNTP. Final concentrations were 200 nM for Polγ-DNA complex, 10 mM for MgCl₂, and 50 μM for dNTP. Reactions were incubated at 37°C for the indicated time and stopped by adding the fourfold excess volume of quench buffer [80% formamide, 50 mM EDTA, 0.1% (w/v) SDS, 5% glycerol, and 0.02% bromophenol blue]. Reaction products were denatured by heating at 95°C for 5 min, resolved on a 20% polyacrylamide denaturing (7 M Urea) gel electrophoresis, and imaged using an Amersham Typhoon Biomolecular Imager (Cytiva).

In vitro deadenylation assays were done as described previously with minor modifications. Deadenylation reactions were carried out at 37 °C in a buffer containing 20 mM PIPES pH 7.0, 40 mM NaCl, 10 mM KCl and 2 mM Mg(OAc)₂. A purified human CCR4–NOT complex (25 nM) was mixed with a synthetic 5′-fluorescein-labeled RNA substrate (50 nM), and the reaction was stopped at the corresponding time point by adding 3× reaction volumes of RNA loading dye (95% (v/v) deionized formamide, 17.5 mM EDTA pH 8 and 0.01% (w/v) bromophenol blue). The reaction products were resolved on a denaturing Tris/borate/ethylenediaminetetraacetic acid–urea polyacrylamide gel, which was subsequently imaged using an Amersham Typhoon Biomolecular Imager (Cytiva).

The right brain hemispheres isolated from mice injected with ¹²⁵I-labeled antibody were cryosectioned sagitally (20 μm) (Cryostar NX70, ThermoFischer) and mounted on Superfrost Plus glass slides (Menzel Gmboltion, Braunschweigh, Germany). Duplicate sections from each animal together with a standard of ¹²⁵I with known radioactivity were exposed to a phosphor imaging plate for 7 days. The plates were scanned in an Amersham Typhoon Biomolecular Imager (Cytiva) with the emission filter IP BP390 at 25 μm/pixel. The generated digital image brightness and contrast were adjusted in ImageJ. The radioactivity standards were used to normalize intensities for images. The whole brain was used as a region of interest for the quantifications in ImageJ.