Immunoprecipitation of c-Jun Protein

Nuclei separation was carried out using a buffer containing sucrose (250 mM) and imidazole (3 mM) pH 7.4, supplemented with protease and phosphatase inhibitors. Cells were scraped from culture dishes and 21 passages were performed through a 22-G syringe. For the recovery of nuclei, lysates were centrifuged at 3400 rpm for 15 min. Then, nucleus fractions (200 μg) were incubated with an anti-c-Jun antibody (1:400) for 2 h at 4 °C. Immune complexes were precipitated with protein G agarose Fast Flow (Millipore) 12 h at 4 °C. Immuno-precipitated proteins were washed 3 times and suspended in Laemmli buffer, separated by SDS-PAGE gels and transferred to PVDF membranes for Western-blot analysis. Protein detection was performed based on a previous protocol [26 (link)].