Mouse anti β actin ac 15

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.