Sc 67

The total cell lysates were prepared with a detergent lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM PMSF; and 1% phosphatase inhibitor cocktail (P2850, Sigma-Aldrich, MO, USA). Western blots were performed as previously reported15 (link) using antibodies of NF1 (sc-67, Santa Cruz, CA, USA); α-tubulin, p-ERK1/2, and ERK1/2 (Cat. #2125, 9101, 9102, respectively; Cell Signaling, MA, USA).

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).