Western blot analysis displaying the reduction of E2F3 expression, induced by E2F3-specific knockdown in 6p22.3-amplified cancer cell line HTB-5. Nonsense RNAi was used as negative control, G3PDH as loading control. Cells were serum starved at the beginning of the experiment (at that time E2F3 is not expressed). Protein was extracted from cell line HTB-5, according to Leone et al. (1998). Ten micrograms protein of each sample was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis for reduced samples on 10% polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Blots were incubated with mouse monoclonal E2F3 Ab-4 primary antibody (1:1000) (Lab Vision, Fremont, CA, USA) followed by incubation with goat anti-mouse IgG secondary antibody (1:2000) (Fc, AP127P; Juro Supply AG, Lucerne, Switzerland). Finally, blots were processed with the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Duebendorf, Switzerland) and exposed to Kodak AR film (Stuttgart, Germany).