Prottot 1kt

Total proteins were extracted from nucleus pulposus samples harvested from rabbit and human discs by grinding in liquid nitrogen (PROTTOT-1KT, Sigma-Aldrich; Merck KGaA). Protein concentrations were then measured using the bicinchoninic acid (BCA) method and the protein expression levels of LIF were detected in each group by western blot analysis. Briefly, 50 µg of extracted proteins were separated by 10% SDS–PAGE, then were electro-blotted onto PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked using 5% blocking buffer (P0252; Beyotime Institute of Biotechnology) at room temperature for 1 h. After incubation with rabbit anti-LIF antibody (1:1,000; SAB2102317; Sigma-Aldrich; Merck KGaA), membranes were washed 3 times with TBST (P0023C; Beyotime Institute of Biotechnology) for 10 min and followed by incubation with a secondary antibody (1:1,000; Sigma-Aldrich; Merck KGaA) for 2 h at room temperature, and were then washed 3 times again with TBST. Relative levels of immunoreactivity were quantified using the Kodak In-vivo Imaging System (Kodak, Rochester, NY, USA). Rabbit anti-GADPH (1:2,000; SAB2108668; Sigma-Aldrich; Merck KGaA) was used as an internal control for the concentration of proteins loaded. Images were analyzed using Image J version 2× (National Institutes of Health, Bethesda, MD, USA).