Ncl p53 cm5p

Six cochleas were dissected from control mice (p53^fl/fl) at P6 and P55, and snap-frozen in liquid nitrogen. Cochleas comprised the otic capsule and the cochlear duct. Tissues were homogenized in lysis buffer (300 mM NaCl, 100 mM Tris-HCl, pH 7.5, 4 mM EDTA, 2% BSA, 0.2% Triton, 2 mM PMSF, 1 mM sodium orthovanadate, Protease Inhibitor Cocktail (Sigma)), incubated on ice for 1 h and centrifuged at 12000 rpm for 15 min at +4 °C. Protein samples (30 μg of each) were resolved by SDS-PAGE and proteins were transferred to a nitrocellulose membrane. Immunoblot analysis was performed using rabbit polyclonal p53 (1:1000, Novocastra; NCL-p53-CM5p) and mouse GAPDH (glyceraldehyde 3-phosphate dehydrogenase, 1:3000, Millipore) antibodies.

In those tumors that were large enough, we froze part of the tumor in liquid nitrogen at the moment of the sacrifice for western blot analysis. Whole-cell protein extracts were subjected to SDS/PAGE using standard techniques. Protein content was determined by the Bradford colorimetric protein assay (BioRad Laboratories; Hercules, CA; USA). The antibodies used in western blots were against p53 (NCL-p53-CM5p, Novocastra, Leica Biosystems, NewCastle, UK); Stat3 (phospho tyr705; 9131), Stat3 (4904); Akt (phospho ser473; 4058) (Cell Signaling Technology, Danvers, MA, USA); p19^ARF (ab80) (Abcam, Cambridge, UK); Akt1/2 (sc-1619), p16^ink4a (sc-1207), Cyclin D1(sc-753), and actin (sc-1616) as a loading control (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Ki67 (1:500, Novocastra), Keratin 10, Keratin 6, Loricrin (1:1,000, Covance), p53 (1:50, NCL‐p53‐CM5p, Novocastra), cleaved‐caspase 3 (1:200, CC3, cs‐96615, Cell Signaling), PCNA (1:200, DAKO), DNMT1 (1:200, Abcam, ab188453), CGAS (1:100 Cell Signaling, 31659; 1:200 Cell Signaling 15102) 5 mC (1:500, Diagenode 006‐500), CD45 (1:1,000, Abcam ab10558), and histone H3S10ph (1:2,000, Santa Cruz Biotechnology SC‐8656‐R).

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).

Snap-frozen tissues from normal brains and tumors were homogenized in 1X SDS loading buffer [50 mM Tris-HCL (pH6.8), 2% SDS, 0.05% bromophenol blue, 10% glycerol, 100 mM β-mercaptoethanol]. Samples were analyzed by SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). The blots were then blocked in 5% nonfat milk in TBST, followed by incubation of primary antibodies at 4 °C overnight. After washing, the blots were incubated in HRP-conjugated secondary antibodies (1706515; 1706516, BioRad) at room temperature for 1 h. Signals were detected using ECL or ECL plus (GE healthcare) followed by film development. The primary antibodies used are as follows: rabbit anti-p53 (NCL-p53-CM5p, 1:1000, Leica Biosystems), rabbit anti-p-Akt(T308) (2965S, 1:1000, Cell Signaling), rabbit anti-p-Akt(S473) (4060L, 1:1000, Cell Signaling), rabbit anti-Akt (9272S, 1:2000, Cell Signaling), rabbit anti-Pten (9559S, 1:1000, Cell Signaling), rabbit anti-p-S6 (5364S, 1:2000, Cell Signaling), rabbit anti-S6 (2217S, 1:2000, Cell Signaling), mouse anti-p120 (Anti-Ras-GAP, 610040, 1:1000, BD Biosciences), rabbit anti-Nf1 (SC-67, 1:1000, Santa Cruz Biotechnology), and mouse anti-β-Actin (A5316, 1:20000, Sigma-Aldrich).