Sc 32748

Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM sodium chloride, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 5mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 1:100 Protease Inhibitor Cocktail (Sigma-Aldrich)) for protein extraction. Proteins were run on 8–12% acrylamide gels, transferred to nitrocellulose membranes and visualized by immunoblotting with the following antibodies anti-ß-ACTIN (1:2000, A2228, Sigma-Aldrich), anti-H-RAS (1:1000, sc-520, Santa Cruz Biotechnology Inc.), anti-MYC (1:1000, AB32072, Epitomics (Abcam)), anti-p16 (1:1000, sc-1207, Santa Cruz Biotechnology Inc.), anti-p19 (1:1000, sc-32748, Santa Cruz Biotechnology Inc.), anti-p53 (1:500, NCL-p53-CM5p, Novocastra), anti-PTEN (1:1000, sc-7974, Santa Cruz Biotechnology Inc.), anti-TSC2 (1:1000, 3990s, Cell signaling) and anti-VINCULIN (1:5000, ab129002, Abcam).

Cell pellets were lysed in Laemmli buffer (100 mM Tris-HCl pH 6.8, 5% glycerol, 2% SDS, and 5% 2-mercaptoethanol). Equal amounts of protein were separated on 12% SDS–polyacrylamide gels and transferred to PVDF(polyvinylidene difluoride) membranes (90 V, 75 min). β-actin was used as a control to ensure equal loading, and images were analyzed using the AlphaView software (ProteinSimple). Immunoblotting was performed using antibodies for MYC (1:1000, Abcam, ab32072), p53 (1:500, Leica Biosystems, NCL-p53-505), p19 (1:250, Santa Cruz Biotechnology, sc-32748), p16 (1:250, Santa Cruz Biotechnology, sc-1207), Axin1 (1:1000, Cell Signaling, #2074), and β-actin (1:10000, Sigma-Aldrich, clone AC-15). Source files of all western blots were provided for Figure 1—figure supplement 2, Figure 4—figure supplement 1, Figure 5—figure supplement 2.