Protein Extraction and Western Blot Analysis

Protein extracts were obtained as in [2 (link), 12 (link), 40 (link)]. Specifically, samples were lysed in buffer containing 50mM Tris-HCl (pH 7.4), 250 mM NaCl, 0.1% tritonX100, 5mM EDTA, 0.3% Empigen BB and supplemented with 1mM PMSF and protease inhibitor mix. Western Blot assay was performed using 40 μg of total extract. Co-immunoprecipitation experiments were performed using 4 μg of antibody for a range of 500-700 μg of protein extract. Ademtech's Bio-Adembeads paramagnetic bead system was used to immunoprecipitate the specific proteins. A negative control was performed with the same amount of protein extract sample immunoprecipitated with the corresponding purified IgG (Santa Cruz). Total proteins were resolved by SDS-PAGE using a 3-8% gradient and/or 10% Invitrogen Precast gel (NuPage, TA or MES buffer). Specific protein signals were revealed with ECL Prime (Amersham, GE Healthcare) and detected by UVIDOC (Eppendorf S.r.l.). The intensity of each band was evaluated by using UVIDOC and/or the NIH Image J 1.8 software (National Institutes of Health, Bethesda, Maryland, USA). Optical density values of specific proteins were normalized to that of tubulin, H1, HSP90 or fibrillarin.