Western Blot Analysis of Protein Samples

A standard procedure was used for preparing cell homogenates, Western 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 [25 (link)]. Filters were incubated with the appropriate primary antibodies overnight at 4 °C, followed by incubation with secondary HRP-conjugated antibodies (goat anti-mouse (cod. 170-6516) or goat anti-rabbit (cod. 170-6515)) 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, Milan, Italy). For loading control, the filters were re-probed with β-ACTIN or GAPDH. Densitometric analysis was performed with Quantity One software (v. 4.5). Original Western Blot data is shown in Supplementary Materials (File S1).