Ecl plus immunoblotting detection system

In order to validate the specificity of the C3-NucView in our model, previously imaged A549 tumor xenografts from mice treated once with intra-tumoral injection of 25μg Cisplatin or 0.05mL saline were explanted at 24h post-treatment. Proteins were extracted, then loaded onto a 15% SDS-polyacrylamide gel, separated and transferred to a nitrocellulose membrane. The membrane was incubated with blocking solution at room temperature for 1h and incubated overnight with primary antibodies against caspase-3 (Cell Signaling Technology, Boston, MA, USA). After incubation with the corresponding HRP-conjugated secondary antibody (Santa Cruz Biotechnology), proteins were visualized using an enhanced chemiluminescence ECL Plus immunoblotting detection system (Amersham biosciences Europe GmbH, Freiburg, Germany).

Protein was extracted by sonication of cells in 100 µL cold 1% SDS containing protease inhibitor (Roche). 40 µg of protein was separated by SDS-PAGE in slab gels of 6% (DNMT1) or 10% (MAT2A) polyacrylamide and transferred to Amersham Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were incubated with primary antibodies overnight at 4°C (DNMT1 [Abcam], MAT2A [Abcam], GAPDH [Santa Cruz]). Antibody binding was revealed by incubation with horseradish peroxidase-conjugated secondary antibodies followed by ECL Plus immuno-blotting detection system (Amersham Biosciences). Chemiluminescence was detected using a FluorChem HD2 camera with FluorChem software (Cell Biosciences). The signals were quantified using NIH Image J64 software and normalized relative to GAPDH.