Ab126171

N2a cells, the ipsilateral cortex of SD rats, and OM-MSCs were processed for western blotting as described previously. Proteins were transferred to the PVDF membrane (Millipore, USA) when the bromophenol blue reached the bottom, and the blots were blocked in 4% BSA in TBST (0.05%) solution for 1 h at room temperature and then incubated at 4°C overnight with the corresponding primary antibody. After incubation with secondary antibodies at room temperature for at least 1 h, the blot was visualized by the ChemiDoc XRS imaging system.
The primary antibodies were as follows: anti-caspase3 (19677-1-AP, Proteintech, USA), anti-GOLPH3 (ab98023, Abcam, Cambridge, UK), anti-SPCA1 (ab126171, Abcam, Cambridge, UK), anti-LC3 (ab192890, Abcam, Cambridge, UK), anti-LAMP1 (ab208943, Abcam, Cambridge, UK), anti-Akt (60203-2-Ig, Proteintech, Chicago, USA), anti-p-Akt (ab38449, Abcam, Cambridge, UK), anti-mTOR (20657-1-AP, Proteintech, Chicago, USA), anti-p-mTOR (ab109268, Abcam, Cambridge, UK), anti-PEDF (bs-20784R, Bioss, Beijing, China), and anti-β-actin (66009-1-Ig, Proteintech, Chicago, USA). The anti-rabbit and anti-mouse IgG secondary antibodies were obtained from Proteintech (Chicago, USA).

The expression of LC3 and SPCA1 was also evaluated by immunofluorescence analysis. After blocking, N2a cells were incubated in the primary antibody against LC3 (14600-1-AP, Proteintech, USA, dilution 1 : 50) or SPCA1 (ab126171, Abcam, Cambridge, UK, dilution 1 : 50) overnight at 4°C. Next, N2a cells were incubated with goat anti-rabbit IgG(H+L) (SA00013-2, Proteintech, USA, dilution 1 : 200) for 90 min at room temperature and then stained with DAPI (Wellbio, Changsha, China). The slides were observed under a fluorescent microscope (Motic, China).