Iκb α antibody

Coimmunoprecipitation assays were performed with a co-immunoprecipitation kit (Pierce, 88804) according to the manufacturer’s instructions, as previously described [28 (link)]. In brief, the spinal dorsal horn tissues were quickly minced and homogenized in lysis buffer. The extracted protein (300 μg) was incubated at 4 °C overnight with IκB-α antibody (1:50, Abcam), and then 20 μL magnetic beads (Millipore, Boston, MA) were added to each sample. The samples were incubated on a 4 °C rotary shaker for 4 h, washed with lysis buffer, and then heated to 96 °C for 10 min with 20 μL of loading buffer to elute the immunocomplexes. The immunocomplexes were then analyzed by western blot as we described.

Proteins were extracted with RIPA buffer. The nuclear extracts were prepared using a NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, USA) according to the manufacturer’s instructions. The protein concentration was determined using a protein assay kit (Bio-Rad). Approximately 30 μg of protein from each sample was separated on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. After being blocked with 5% skim milk, the membranes were incubated with IκBα antibody (Abcam Cambridge, UK), p-IκBα antibody (Abcam Cambridge, UK) and p65 antibody (Abcam Cambridge, UK) overnight at 4 °C, followed by incubation with the corresponding secondary antibodies for 1 h at room temperature. Proteins were detected using Super ECL Plus Detection Reagent (Thermo Fisher Scientific, Carlsbad, CA, USA). β-actin was used as an endogenous control for total and cytosolic extracts. As for NF-κB nuclear p65, Lamin B served as the nuclear loading control.