Supplemental Methods
Western Blotting. Western blots were immunostained for AKT-HA using an anti-HA monoclonal antibody (HA.11, Covance, Berkely, CA) at a 1:1000 dilution; BRAFV600E-V5 using a mouse monoclonal antibody targeting V5 (R960-25, Invitrogen, Carlsbad, CA) at a 1:1000 dilution; KRASG12D using a pan-RAS antibody (05-516, Millipore, Temeculah, CA) diluted 1:1000; CRAF using a RAF-1 antibody (sc-133; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200, and anti-α-tubulin (T9026, Sigma, St. Louis, MO) at a 1:5000 dilution. MEK1/2 (9122, Cell Signaling, Boston, MA) and Phospho-MEK1/2 (Ser221) (2338, Cell Signaling, Boston, MA) antibodies were used at a 1:200 dilution. Detection of MAPK activation was performed using a 1:1000 dilution of a rabbit monoclonal antibody directed against phosphorylation of ERK at Thr202/Tyr 204 (4370, Cell Signaling, Boston, MA). Detection of total ERK protein levels was performed using a 1:5000 dilution of anti-ERK1/2 antibody (9102, Cell Signaling, Boston, MA). Detection of AKT activation was performed using a 1:1000 dilution of a rabbit monoclonal antibody directed against phosphorylation of AKT at Ser473 (4060, Cell Signaling, Boston, MA). Detection of total AKT protein levels was performed using a 1:1000 dilution of anti-AKT antibody (9272, Cell Signaling, Boston, MA). The blots were then incubated with an anti-mouse or rabbit IgG-HRP secondary antibody as appropriate (Cell Signaling, Boston, MA) for 1 h at room temperature. The blots were incubated with ECL per the manufacturer’s specifications (Amersham, Piscataway, NJ), and exposed to film.
Immunohistochemistry (IHC). Antibodies or antisera against the following antigens were used for IHC: GFAP (Z0334; 1:500; DAKO, Carpinteria, CA); Olig-2 (AB33427-100; 1:100; Abcam, Cambridge, MA); MAP-2 (MAB3418; 1:500; Chemicon, Billerica, MA); doublecortin (AB18723; 1:500; Abcam, Cambridge, MA); synaptophysin (080130 prediluted; Zymed, San Francisco, CA); CD15 (559045; 1:1000, PharMingen San Diego, CA); AKT (detected using an antibody to the HA epitope: HA.11; 1:1000; Covance, Berkeley, CA); and nestin (ab6142,1:200; Abcam, Cambridge, MA). Sections were incubated with biotinylated secondary antibodies and a horseradish peroxidase-conjugated streptavidin complex. Visualization was carried out with DAB and sections were counterstained with hematoxylin.