Anti nras

Slides with cryosections were dried up at room temperature and then boiled in the boiling buffer (800 ml milli-Q water, 4 ml 1 M Tris pH8, 1.6 ml 0.5 EDTA) for antigen recovery. Then the brain sections were blocked in 150 μl blocking buffer (0.1 M PBS; 10% NGS and 0.1% Tween 20) and incubated overnight with the following primary antibodies: 1:300 Anti-Nras, Abcam, ab77392; 1/500 Anti-GFP, Proteintech, 50430-2-AP; 1/500 Anti-mCherry, Abclonal, AE002. Signals were visualized using the 1:400 diluted secondary antibody conjugated with AlexaFluor-488, -561 or −647 (Proteintech and Abclonal). After nucleic DNA staining by 4′,6-diamidino-2-phenylindole (DAPI, Sigma), slices were mounted with fluorescent anti-fade mounting medium (Dako). Images were acquired using the Ni-E A1 HD25 confocal microscope (Nikon).

The following antibodies were used at the dilutions specified—immunofluorescent (IF) or Western blot analysis (WB): anti-Arl13b, 1:2000 IF (clone N295B/66, UC Davis/NIH NeuroMab Facility), anti-Ac3 (gift from Young-Goo Han laboratory), anti-Siah2, 1:200 IF (Santa Cruz Biotechnology, cat. no. SC81787); anti-Tag1, 1:50 IF (UC Davis/NIH NeuroMab Facility, cat. no. 4D7/TAG1); anti-β-actin, 1:20000 WB (Sigma, cat. no. A2228); and anti-phospho-Mek1/2, 1:500 IF (Cell Signaling, cat. no. 9121); anti-H-Ras, 1:500 WB (Abcam, cat. no. ab32417); anti-N-Ras, 1:500 WB (Abcam, cat. no. ab77392); anti-K-Ras, 1:2000 WB (Proteintech, cat. no. 12063-1-AP); anti-vitronectin, 1:200 IF (Thermofisher cat. no. MA5-24083); anti-integrin β1 (Biolegend. Clone HMβ1-1); anti-myc 1:2000 WB (Thermofisher, cat. no. PA1-981); anti-HA 1:2000 WB (Biolegend, clone 16B12).