Typhoon 9400 system

Northern blotting was performed as described previously (47 (link)), except that cells were lysed by bead beating with 0.5-mm-diameter glass beads (no. 11079105z; Stratech Scientific, Newmarket, United Kingdom) in a Precellys 24 instrument (Bertin Technologies, Montigny-le-Bretonneux, France) at 4°C with two cycles of 30 s at 6,500 rpm, separated by a 30-s break. The probes for HAC1 (47 (link)), KAR2 (52 (link)), PDI1 (52 (link)), and the loading control, pC4/2 (53 (link)), were described previously. All mRNAs were quantified by phosphorimaging on a Typhoon 9400 system (GE Healthcare, Little Chalfont, United Kingdom). Volumetric measurements were normalized to the loading control, pC4/2. The percentage of HAC1ⁱ is defined as 100 × HAC1ⁱ/(HAC1^u + HAC1ⁱ + 1st exon and intron + 1st exon), and the percentage of HAC1 cleavage is defined as 100 × [HAC1ⁱ + 0.5 × (1st exon and intron + 1st exon)]/(HAC1^u + HAC1ⁱ + 1st exon and intron + 1st exon).

Primer extension reactions with linear or circular templates were performed in a buffer containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl₂ and 1 mM DTT. 5 nM radiolabeled DNA substrate was added to the reaction mixtures (10 μL total volume) as well as dNTPs at 3 different concentrations: “low” (2 μM dTTP, 1 μM dCTP, 2 μM dATP and 1 μM dGTP), “normal” (10 μM dTTP, 5 μM dCTP, 5 μM dATP and 3 μM dGTP) or “high” (200 μM equimolar). The reactions were performed at 37 °C and started by addition of 200 nM PrimPol. At the indicated time points, the reactions were stopped by the addition of 1.1 μL of termination mixture (5% SDS, 250 mM EDTA) and analyzed on a 8–12% polyacrylamide gel containing 8 M urea. Quantification was performed by phosphorimaging of the dried gel (³²P) on a Typhoon 9400 system (GE Healthcare).

The XO44 probe (Sigma, PF-6808472) was added to live PLC/PRF/5 cells and Huh7 cells at a final concentration of 2 µM followed by incubation for 30 minutes at 37 °C. Cell pellets were collected by centrifugation at 300 × g at 4 °C and resuspended in lysis buffer containing 100 mM HEPES, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF, and 1× complete EDTA-free protease inhibitor cocktail (Roche). Cellular debris was removed by centrifugation at 15,000 × g for 30 minutes at 4 °C. Cell lysates were used for the click reaction via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Click chemistry was initiated by sequential addition of the following to each lysate: 1% SDS, 100 µM TAMRA-azide or biotin-azide, 1 mM TCEP, 100 µM TBTA, and 1 mM CuSO. After 1.5 hours of incubation at room temperature, the reactions were quenched by acetone precipitation. The precipitated proteins were pelleted by centrifugation at 1000 × g at 4 °C for 10 minutes, and the pellets were resuspended in sample buffer and resolved by 12% SDS-PAGE. Gels were scanned by a Typhoon 9400 system (Typhoon Imaging System, GE) under the Cy3/Cy5 channel for fluorescence bands and protein marker visualization. The gels were then stained with Coomassie blue to show the protein loading amount.