Rabbit antibody against cleaved active caspase 3

According to the time node of the experiment, the whole cell protein was extracted. The method follows: After washing PBS three times, add precooled Lysis cracking solution (100 μL/well), carefully scrape the cells and thoroughly mix the lysate with the cells. Transfer the suspension into the precooled EP tube, ice bath for 30 min and centrifugation at 4 °C for 15 min, 12,000 rpm. The total protein concentration of the tissues or cells was analysed with a BCA kit (Sigma, CA, USA). Rabbit antibody against cleaved (active) caspase-3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal antibodies against Bcl-2 or Bax (1:1,000, Santa Cruz, CA, USA) and rabbit antibody against PriB (1:1,000, Abcam, USA), and β-action (1:2,000, Anbo, USA). Subsequently, the blots were probed with horseradish peroxidase (HRP)-conjugated goat secondary antibody against rabbit or mouse IgG (1:1,000, Abcam, USA). Detection and quantitation were performed with a Typhoon 9400 Variable Mode Imager (GE Healthcare) and Lumi-Light Western Blotting Substrate (Roche Diagnostics) for HRP-labelled blots.

We analyzed the expression of Ngn2 NGF, BDNF, phosphorylation TrkB (p-TrkB), total TrkB, cytochrome C (CytC) in the cytoplasm (C CytC), CytC in the mitochondria (M CytC), active caspase 3, Bcl-2 and Bax by western blotting in vivo and in vitro as described previously. The total protein concentration of the tissues or cells was analyzed with a BCA kit (Sigma, CA, USA). The blots were probed with mouse monoclonal anti-Ngn2 rabbit antibody (1:200; Abcam, USA), mouse monoclonal antibody anti-6-His (1:1,000; Abcam, USA), monoclonal anti-NGF (1:2,000, Abcam, USA), mouse monoclonal anti-BDNF (1:2,000, Abcam, USA), rabbit monoclonal anti-p-TrkB or TrkB (1:1,000, Affinity, USA), rabbit polyclonal anti-CytC (1:1,000, Abcam, USA), rabbit antibody against cleaved (active) caspase-3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal antibodies against Bcl-2 or Bax (1:1,000, Santa Cruz, CA, USA), Cytochrome C oxidase IV (COX IV) and β-actin (1:2,000, Anbo, USA). Subsequently, the blots were probed with a horseradish peroxidase (HRP)-conjugated goat secondary antibody against rabbit or mouse IgG (1:1,000, Abcam, USA). Detection and quantitation were performed with a Typhoon 9400 Variable Mode Imager (GE Healthcare) and Lumi-Light Western Blotting Substrate (Roche Diagnostics) for HRP-labeled blots.