Rabbit anti e2f1

Samples were lysed in ice in RIPA buffer (1% NP-40, 150 mM NaCl, 5 mM EDTA, 0.25% NaDOC, 50 mM Tris-HCl pH 7.5, SDS 0,1%) added with protease and phosphatase inhibitors, sonicated to shear DNA and centrifuged at 14000 r.p.m. for 20 min at 4 °C; supernatant was collected as WCE. For co-immunoprecipitation experiments 700 μg WCE were diluted with IP buffer (0.5% NP-40, 100 mM NaCl, 5 mM EDTA, 10% glycerol, 50 mM Tris-HCl pH 7.5) added with protease and phosphatase inhibitors to a final volume of 450 μl and incubated overnight at 4 °C with Dynabeads Protein G (Life Technologies) pre-conjugated with anti-iASPP antibody (49.3, Santa Cruz Biotechnology) or irrelevant IgG (Life Technologies). Beads were washed three times with IP buffer, proteins were eluted with Laemmli buffer and visualized on SDS polyacrylamide gel electrophoresis. The following antibodies were used for western blot: rabbit anti-iASPP (ab34898), (Abcam, Cambridge, United Kingdom), rabbit anti-E2F1 (#3742), rabbit anti-BCL2 (#2976), mouse anti-GLI1 (L42B10) (Cell Signaling Technology, Danvers, MA, USA), goat anti-GLI2 (#AF3635; R&D Systems), mouse anti-Myc (9E10), mouse anti-HSP90 (F-8), mouse anti-p53 (DO-1), mouse anti-iASPP (2808C5a), rabbit anti-CDK1 (C-19), rabbit anti-Cyclin B1 (H-433; Santa Cruz Biotechnology), mouse anti-β-ACTIN (AC-15; Sigma-Aldrich, St. Louis, MO, USA). Chemiluminescent detection was used.

For IHC analysis, the tissue sections were deparaffinized and rehydrated. Endogenous peroxidase activity was inhibited with 0.3% H₂O₂ in methanol. For antigen retrieval, the slides were boiled in 0.01 M (pH 6.0) sodium citrate buffer for 15 min. After blocking with 5% normal goat serum, the slides were incubated with primary antibodies (rabbit anti-SET7/9, 1:50; cat. no. 2813; Cell Signaling Technology, Inc., Danvers, MA, USA; rabbit anti-E2F1, 1:1,000; cat. no. ab179445; Abcam, Cambridge, MA, USA) at 4°C overnight. Subsequently, biotinylated secondary antisera were applied and the slides were incubated with streptavidin-biotin horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 30 min at room temperature. Finally, the visualization signal was developed by incubation in 3–3′-diaminobenzidine solution and hydrogen peroxide (Maixin, Fuzhou, China), followed by counterstaining with Mayer's haematoxylin. Images were taken (magnification, ×40) with an Olympus BX53 light microscope (Olympus Corp., Center Valley, PA, USA).