Anti iκb α antibody

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)}.

Protein from kidney tissues was extracted with RIPA lysis buffer, and Western blot analysis was performed as described recently [22 (link)]. Protein concentration was determined using the BCA protein assay (Sigma–Aldrich). Fifty micrograms of proteins were loaded in each lane of a SDS/PAGE (10% gel) and run at 100 V. Protein were transferred to nitrocellulose membranes, which were then washed and incubated with blocking buffer (5% non-fat milk in PBS containing 0.1% Tween 20) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with the primary antibodies: collagen type I (Col I) (Abcam rabbit polyclonal, 1:1000), fibronectin (Fn) (Sigma–Aldrich chicken polyclonal, 1:1000), plasminogen activator inhibitor-1 (PAI-1), IL-6 (Santa Cruz Biotechnology rabbit polyclonal, 1:1000), ED-1, ICAM-1 (Santa Cruz Biotechnology mouse monoclonal, 1:1000), MCP-1 (Santa Cruz Biotechnology goat polyclonal, 1:1000), anti-IκB-α antibody (Abcam rabbit monoclonal, 1:200) and β-actin (Sigma–Aldrich mouse monoclonal, 1:2000). After three washes, the membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature followed by three washes with TBST. Antibody labelling was visualized by the addition of chemiluminescence reagent (Amersham Biosciences), and the membrane was exposed to Kodak XAR-5 film.

All of the chemicals used in this study were obtained from Sigma (St. Louis, MO, USA) unless otherwise indicated. Anti-Aurora-A, anti-phospho-Aurora-A (Thr288), anti-caspase 3, and anti-phospho-IκBα (Ser32-36) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-IκBα antibody was obtained from Abcam (Cambridge Science Park, Cambridge, UK). Anti-PLCG1, anti-PIK3R1, and anti-GAPDH antibodies were obtained from Abnova Corporation (Taipei, Taiwan). Anti-NF-κB p65 and anti-MALT1 antibodies and anti-rabbit and anti-mouse IgG-conjugated horseradish peroxidase secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).