Versadoc mp 400 system

Circular DNA molecules were analyzed in neutral/neutral two-dimensional agarose gels, a technique that allows to separate DNA molecules by mass and shape. The first dimension was run at 1.5 V/cm in a 0.4% agarose (Seakem; FMC Bioproducts) gel in Tris-borate-EDTA (TBE) buffer for 17-19 h at room temperature. The second dimension was run at 7.5 V/cm in 1% agarose gel in TBE buffer for 7-9 h at 4°C. Chloroquine (Sigma) was added to the TBE buffer of both the agarose gel and the running buffer. After electrophoresis, gels were subjected to Southern hybridization. A 177-pb probe was obtained by PCR amplification of the kanamycin-resistance gene of plasmid pSUM36 (Ainsa et al., 1996 (link)) with oligonucleotides PSUM36FBIOT 5′-TCGGCAGGAGCAAGGTGAGATGA-3′ (biotinylated at 5′) and PSUM36R 5′-CCTGTCCGGTGCCCTGAATGAA-3′. This probe was used on the nylon membranes (Inmobylon NY⁺, Millipore) onto which the two-dimensional agarose gels had been transferred. Chemiluminescent detection of DNA was performed with the Phototope^® -Star kit (New England Biolabs). Images were captured in a VersaDoc MP400 system and analyzed with the Quantity One program (Bio-Rad).

Frozen tissue samples were lysed in RIPA lysis buffer on ice, and protein was isolated. Protein quantification was done by BCA assay (Thermo Scientific, USA). Fifty micrograms of protein was loaded on a 10% polyacrylamide gel for electrophoresis. Proteins were blotted to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After blocking in 3% bovine serum albumin (BSA), incubation with primary antibodies was done overnight at 4°C. The following antibodies were used: pERK (44/42 kDa, 9102S), ERK1/2 (44/42 kDa, 9106S, both from Cell Signaling Technologies, USA). 14-3-3 served as loading control (28 kDa, K-19, Santa Cruz Biotechnology, USA). After washing in PBS horseradish peroxidase (HRP) conjugated secondary antibodies (Dianova, Hamburg, Germany) were incubated for 1 h at room temperature. Proteins were visualized by incubation with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA). To enhance signal intensity SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA) was used according to the manufacturer's instructions. Bands were immediately captured by VersaDoc MP 400 System (Bio Rad, USA). Five mice per group were analyzed.