Anti pten antibody

Western blot analysis were conducted following standard protocols as described earlier [42 (link)]. Briefly, whole cell lysates were prepared and protein concentrations were quantified colourimetrically using a BCA Protein Assay Kit (Solarbio, Beijing). Samples were separated in a 10% SDS-polyacrylamide gel (Solarbio, Beijing) and blotted onto a PVDF membrane (Millipore). Immunoblot was performed with commercially available antibodies (anti PTEN antibody, 1:5000, Santa Cruz; anti- AKT antibody, 1:1000, BD Bioscience; anti-P-AKT antibody, 1:1000, Cell Signaling Technology; anti-GAPDH antibody, 1:5000, Santa Cruz.). ECL (Millipore) was applied for chemiluminescence detection. Immunoblot signal quantifications were performed using Image J software.

The total proteins from L_4–6 spinal cords or VSC4.1 neurons were extracted and purified with a protein extraction kit (KangChen, China) [4 (link), 6 (link)]. After determination by a BCA protein assay kit (Beyotime Biotechnology, China), equal protein concentrations were loaded onto a 10% SDS-PAGE gel and transferred to PVDF membranes. The membranes were incubated with 5% skim milk for 1 h to avoid nonspecific binding and probed with an anti-calpain-1 antibody (Santa Cruz Biotechnology, sc-271313, 1 : 400, Dallas, USA), anti-calpain-2 antibody (1 : 500), anti-p35 (1 : 400), anti-TPPP/p25 antibody (1 : 500), anti-PTEN antibody (Santa Cruz Biotechnology, sc-7974, 1 : 400, Dallas, USA), anti-Cdk5 antibody (Santa Cruz Biotechnology, sc-6247, 1 : 300, Dallas, USA), anti-caspase-8 p18 antibody, anti-caspase-3 antibody (Abcam, ab184787, 1 : 500, CA, USA), or β-actin (Santa Cruz Biotechnology, sc-47778, 1 : 2000, Dallas, USA) overnight at 4°C. After washing, the membranes were incubated with peroxidase-conjugated secondary antibodies (Beyotime Biotechnology, A0192, 1 : 10,00, China) for 2 h at room temperature. The blots were detected by an ECL kit (Beyotime Biotechnology, China) and quantified by Quantity One software (Bio-Rad Laboratories, Italy).