Anti epor

Estimation of the Epo and EpoR protein expression was performed by western blot analysis. Total lysis of the cells and spinal cords was conducted using the Radio-Immunoprecipitation Assay buffer (Thermo Fisher Scientific, Inc.), and protein concentrations were determined using the bicinchoninic acid protein assay (Thermo Fisher Scientific, Inc.). Equal amounts (20 µg) of the proteins were separated by SDS-PAGE gel, which were subsequently electroblotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk and were probed overnight at 4°C with the primary antibodies anti-Epo (1:800; Santa Cruz Biotechnology, Inc.) and anti-EpoR (1:800; Santa Cruz Biotechnology, Inc.). The membrane was then incubated with secondary horseradish peroxidase-conjugated antibody (1:2,000; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. The membranes were then prepared using an electrochemiluminescence western blotting kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The optical density value of each band was quantified using ImageJ and was normalized to the corresponding β-actin level. Values were expressed as the fold change relative to the control value.

Twenty-four micrograms of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently blotted onto a polyvinylidene fluoride (PVDF) membrane. The transferred membranes were blocked in 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and then incubated with primary antibodies overnight at 4°C. Anti-EPO and Anti-EPOR were obtained from Santa Cruz Biotechnology, and the anti-neuron-specific enolase (anti-NSE) antibody was purchased from Abcam (Cambridge, MA, USA). Following incubation with the primary antibody, the membranes were rinsed and incubated with the HRP-conjugated anti-rabbit secondary antibody and developed using a standard enhanced chemiluminescent (ECL) system, and the signal was captured by an Amersham Imager 600 (GE Healthcare, Madison, WI, USA). All of the procedures were performed in triplicate.