Hybond n nitrocellulose membrane

Total RNA was prepared in the same way as for the EMOTE assay, but with an additional step of ethanol precipitation to concentrate the RNA. 4 µg total RNA from each strain was loaded on a 5% polyacryl amide gel with 8M urea, and afterwards transferred to a Hybond-N nitrocellulose membrane (Amersham) using a Biorad Protean Tetra-cell blotting system. The RNA was crosslinked to the membrane with a UV Stratalinker 2400 (Stratagene), and the marker was revealed using Methylene blue. Probes were hybridised over night at 37°C in ExpressHyb hybridization solution (Clontech, Mountain View, CA, USA), excess probe was washed away, and the signal was detected using a Typhoon FLA 7000 phosphorimager (General Electric). The membrane was stripped for 2 hours at 75°C with 0.2% SDS and 10 mM Tris pH 7.5, whereupon a new probe was hybridised. Loading control was done with a 5S rRNA probe, used last to ensure that the strong 5S signal did not interfere with the other experiments. Probes used for Northern blotting (Table S9) were 5′ labelled using ³²P γ-ATP and T4 polynucleotide kinase (New England Biolabs).

Cells were lysed with 10 mM Tris-HCl (pH 8.5), 2 mM MgCl₂, and 10% SDS solution following 10 mM MMS (Sigma) treatment for 30 min. When lysed with PARG inhibitor, 1 µM ADP-HPD and 5 µM GLTN were included. Next, the samples were incubated 2 h with additional 0.1% proteinase K (Thermo Scientific). The samples were then extracted with equal volumes of phenol/chloroform and chloroform, and the aqueous layer was obtained. PAR was recovered from the aqueous layer with ethanol precipitation by adding 0.1 volumes of 3 M NH₄Ac (pH 9.0) and 2 volumes of ethanol at room temperature. After centrifugation at 16,000 × g for 30 min, the pellet was washed with 70% ethanol and dried, and the pellet was resuspended into 20 µl deionized water for dot blotting.
Purified PAR was dotted on Hybond-N+ nitrocellulose membrane (Amersham Pharmacia Biotech). After drying at 55 °C, the membrane was blocked with 10% non-fat milk for 1 h at room temperature followed by 2-h incubation with anti-PAR antibodies at room temperature. After three consecutive 10-min washes with TBST, the membrane was incubated with horseradish peroxidase-conjugated goat-anti-rabbit secondary antibody for 1 h. The membrane was washed again for three times with TBST and developed using the Enhanced Chemi-Luminescence plus (ECL+) detection system (GE Healthcare).

Mouse skin biopsies were incubated overnight at 65 °C in DirectPCR Lysis Reagent (Euromedex) and 2% proteinase K (Sigma). DNA was extracted by using sodium acetate/ethanol precipitation and quantified on a Nanodrop spectrophotometer. 500 ng of genomic DNA were mixed with 1% SYBR Green (Brilliant III Ultra-Fast SYBR^®), dot-blotted onto a Hybond N+ nitrocellulose membrane (Amersham) and dried at 80 °C for 30 min. Membranes were blocked for 20 min (20 mM TBS, 5% non-fat dry milk, 0.5% Tween 20, pH 7.6) and incubated with anti-CPD monoclonal antibody (1:1000, Kamiya Biomedical) overnight at 4 °C. Membranes were washed in TBS and incubated for 1 h with an HRP-conjugated secondary antibody (1:2000, Vector Laboratories). Blots were developed using an ECL reagent (Biorad). Chemiluminescence signals were quantified and normalized against SYBR green fluorescence.