Total cell extracts were obtained by lysing the cells directly using 2× SDS-PAGE sample buffer. Western blotting and processing were then performed as described previously [35 (link)]. The following antibodies were used: mouse monoclonal anti-human pRB (BD Pharmingen); rabbit polyclonal anti-MMP2 and MMP9 (Abcam); rabbit monoclonal p21 (12D1), rabbit polyclonal phospho-Akt (Ser473), rabbit monoclonal pan Akt (Cell Signaling), mouse monoclonal anti-p53 (DO-1), mouse monoclonal anti-β-actin, mouse monoclonal anti-SAP97 (2D11) (Dlg), rabbit polyclonal p130 (C-20) and p107 (C-18), mouse monoclonal p-ERK1/2 (12D4), and ERK1/2 (C-9) were from Santa Cruz.
Immunoblots were developed using Clarity™ Western ECL Substrate (Bio-Rad) and images were captured using the ChemiDoc™ Imaging System (Bio-Rad). Protein band intensities were quantified using ImageJ and normalized with the levels of β-actin, which serves as a loading control.
Immunoblots were developed using Clarity™ Western ECL Substrate (Bio-Rad) and images were captured using the ChemiDoc™ Imaging System (Bio-Rad). Protein band intensities were quantified using ImageJ and normalized with the levels of β-actin, which serves as a loading control.