Ab46172

The neurons were lysed using RIPA buffer (#9806, Cell Signaling Technology, Danvers, MA, USA). Protein concentration was determined with a Bicinchoninic protein assay (BCA) kit (7780S, Cell Signaling Technology, Danvers, MA, USA). All the protein samples were then electrophoresed via 10% SDS-PAGE (P0012A Beyotime, China). Later, the samples were transferred to polyvinylidene fluoride membranes (PVDF; Millipore, Bedford, MA) which were blocked by 5% skimmed milk at room temperature for 60 min. The membranes were then incubated with the following primary antibodies overnight at 4ºC: anti-GAPDH antibody (rabbit, #5174, 1 : 1000, Cell Signaling Technology, USA), anti-NGF antibody (rabbit, ab221609, 1 : 1000, Abcam, UK), anti-C-caspase-3 antibody (rabbit, ab2302, 1 µg/ml, Abcam, UK), anti-CNTF antibody (rabbit, ab46172, 1 : 3000, Abcam, UK) and anti-BDNF antibody (rabbit, ab108319, 1 : 1000, Abcam, UK). The membranes were then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-rabbit IgG H&L (HRP) (ab205718, 1 : 2000, Abcam, UK) for 1 h at room temperature and washed with tris-buffer saline tween (TBST) three times. GAPDH was employed as the normalization reference. An enhanced chemiluminescence (ECL) plus kit (K22030, Abbkine Scientific, China) was employed to measure the labelled proteins [36] .