Quantitative Western Blot Analysis

Cells were seeded in T25 flasks and treated when confluence reached approximately 80%. A standard procedure was used to prepare cell homogenates, SDS-PAGE and blotting onto a PVDF filter (cod. 162-0177; BioRad, Hercules, CA, USA), and the saturation of the membrane with non-fat dry milk, as detailed in [28 (link)]. Filters were incubated with the appropriate primary antibodies overnight at 4 °C, followed by incubation with secondary HRP-conjugated antibodies for 1 h at room temperature. The bands were detected with Enhanced Chemiluminescence reagents (ECL, cod. NEL105001EA; Perkin Elmer, Waltham, MA, USA) and imaged with the VersaDOC Imaging System (Universal Hood II—S.N. 76S/04219; Biorad, Hercules, CA, USA). For loading control, the filters were re-probed with β-tubulin, β-actin, or histone H3. Densitometric analysis was performed with the Quantity One software (v. 4.5).