Gel drier

Freshly isolated untreated CD-1 mouse liver samples were homogenized in cold Dulbecco’s PBS (DPBS) containing protease inhibitor cocktail (Roche) with a glass/Teflon homogenizer. Homogenates were centrifuged at 4°C for 20 min at 21 000× g to remove insoluble cellular debris. Next, the protein-containing supernatant was transferred to fresh tubes, and protein concentration was determined by the BCA assay (Pierce). Protein concentrations were adjusted to 4 mg/ml with DPBS and glycerol at 1:0.6 (lysate:glycerol) ratio. Samples were aliquoted into single dispense tubes and then stored at −80°C. LNA-modified ASOs were 5′-end labeled with [γ ³²P]ATP (6000 Ci/mmol, 10 mCi/mL, Perkin Elmer) and T4 polynucleotide kinase with the KinaseMax™ kit (Ambion) according the supplied protocol with one modification. Binding reactions were set up in 20 µl reactions by mixing of 30 000 cpm (∼1–5 pmol) of LNA compounds with 40 µg of prepared liver lysate. The reactions were incubated for 30 min at room temperature and subsequently resolved by 6% non-denaturing polyacrylamide gel electrophoresis. Gels were dried using a BioRad gel drier and exposed on phosphor imaging screens overnight. The phosphor screens were scanned and quantified using the Perkin Elmer Cyclone Plus® storage phosphor system and associated software.

Oligonucleotides were 5′-end labeled using [γ-³²P] ATP (6000 Ci/mmol) and T4 polynucleotide kinase, purified by 19% denaturing polyacrylamide gel in 1× TBE, visualized by storage phosphor autoradiography, excised, and extracted by crush and soak. The labeled oligonucleotide was hybridized to its unlabeled complement by heating to 95°C for 5 min in 1× TE and 200 mM NaCl, followed by slow cooling to room temperature. Samples containing less than 1.1 nM RNA and different concentrations of hADAR2-R2D mutants (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0 nM) were equilibrated in 20 mM Tris–HCl, pH 7.0, 3.5% glycerol, 0.5% DTT, 60 mM KCl, 20 mM NaCl, 0.1 mM BME, 1.5 mM EDTA, 0.003% NP-40, 160 units/ml RNasin, 100 μg/ml BSA, and 1.0 μg/ml yeast tRNA for 30 min at 30°C. Samples were loaded onto a running 6% nondenaturing polyacrylamide gel (79:1 acrylamide:bisacrylamide) and electrophoresed in 1× TBE buffer at 4°C for 90 min. The gels were dried on a gel drier (Biorad) for 60 min at 80°C under vacuum followed by exposure to storage phosphor imaging plates (Kodak) for 3 h in the dark. After exposure, the dried polyacrylamide gels were removed, and the phosphor imaging plates were scanned by Typhoon Trio Variable Mode Imager (GE Healthcare). See Supplementary Table S2 for RNA sequences.

Engineered gfp-tmfA P. pastoris (1.5 × 10⁸ cells) were pelleted at 14,000 rpm for 5 min and lysed by vortexing in 50 mM Tris–HCl, pH 7.9, 2% SDS, 5% β-mercaptoethanol using glass beads for 30 min at 4°C. The mixture was heated for 5 min at 90°C, centrifuged at 14,000 rpm for 5 min at room temperature and supernatants collected. Aliquots (80 μl) were removed and mixed with 20 μl of sample buffer [0.5 M Tris–HCl, pH 6.8, glycerol 10% (v/v), 25% β-mercaptoethanol, and 0.05% Bromophenol blue]. The extracted proteins were separated by SDS-PAGE on a slab poly acrylamide gel (Laemmli, 1970 (link)) and stained with Coomassie Blue R-250 and distained in MeOH acetic acid for 18 h. Distained gels were then dried using a gel drier at 70°C (BioRad, CA, United States).