Anti mcherry

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 purchased from Cell Signaling Technology: anti-IRS-1 (2382), anti-pAKT S473 (9271), anti-p85 (4292), anti-IGF-1Rβ (9750), anti-PDK1 (13037). Other antibodies were from the following commercial sources: anti-VAPB (Sigma-Aldrich, HPA013144), anti-NOGOA (Bio-Rad, AHP1799), anti-BAP31 (Santa Cruz Biotechnology, sc-48766), anti-PERK (abcam, ab229912), anti-pPERK (Affinity Biosciences, DF7576), anti-AKT (HUABIO, ET1609-47), anti-Actin (HUABIO, M1210-2), anti-Tubulin (HUABIO, M1305-2), anti-GFP (HUABIO, ET1607-31), anti-FLAG (YEASEN, 30503ES60), anti-HA (Invitrogen, PA1-985) and anti-mCherry (ABclonal, AE002).