Analysis of IκB-α Protein Levels

Western blotting was performed essentially as described^{53 (link),59 (link)}. Confluent HAoSMCs (1.2 × 10⁶ cells) deprived of serum for 16 h were incubated with SBSN_HUMAN[225–237] or SBSN_HUMAN[243–259] for the indicated time, washed three times with phosphate-buffered saline (PBS) and solubilized in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) containing 10 μM protein inhibitors (Thermo Fisher Scientific). Extracted protein samples were loaded onto 4–20% gradient polyacrylamide gel (Bio-Rad Laboratories) and transferred to PVDF membranes (Immune-Blot or Trans-Blot Turbo™ Mini PVDF Transfer Packs, Bio-Rad). After blocking with Blocking One (Nacalai Tesque), membranes were incubated with an anti-IκB-α antibody (1:3000, Abcam, Cambridge, UK) or an anti-β-actin antibody (1:3,000, Abcam) overnight at 4 ℃, extensively washed, and incubated with a peroxidase-coupled secondary antibody (1:10,000, BioRad) for 1 h at room temperature. Protein bands were detected using an enhanced chemiluminescence system (ECL prime, GE Healthcare). The signals of each blot were visualized and quantitatively analyzed using ImageQuant LAS 4000 (GE Healthcare)^{54 (link)}.