Af3010

Kidney tissues were homogenized and lysed in RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (25 μg) were separated using SDS-PAGE and transferred to PVDF membranes (Beyotime). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti-IRAK2 (1 : 1,000; #DF4782, Affinity, MI, USA), anti-p-NF-κB (1 : 1000; #AF2002, Affinity), anti-NF-κB (1 : 1,000; #AF5006, Affinity), anti-p-IKKβ (1 : 1,000; #AF3010, Affinity), anti-IKKβ (1 : 1,000; #AF6009, Affinity), and anti-GAPDH (1 : 5,000; #AF7021, Affinity). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (1 : 5,000; #S0001, Affinity) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific) and quantified using the ImageJ software (National Institutes of Health, MD, USA).