Protein Expression Analysis by Western Blotting

The cells were lysed after the 24 h treatment and the proteins were separated using electrophoresis, as previously described [38 (link)]. The primary antibodies were: rabbit anti-p21 (1:1000, Cell Signaling Technology, distributed by Euroclone, Milan, Italy), rabbit anti-phospho Rb (1:1000, Cell Signaling Technology) and rabbit anti Rb (1:1000, Cell Signaling Technology). The membrane was washed in a T-PBS buffer, then incubated for 1 h at RT with a goat anti-rabbit IgG Alexa Fluor 750 antibody or with a goat anti-mouse IgG Alexa Fluor 680 antibody (Invitrogen, Milan, Italy) and then visualized using an Odyssey Infrared Imaging System (LI-COR^® Bioscience, distributed by Carlo Erba, Milan, Italy). The rabbit anti-vinculin (1:1000, Cell Signaling Technology) was used in order to assess the equal amounts of protein loaded in each lane.