Anti phosphorylated p65

Following the treatment of podocytes with 6.25, 12.5 or 25 µM BA, total protein was extracted from the conditionally immortalized mouse podocytes using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and 30 µg protein/lane was separated by 10% SDS-PAGE. The separated proteins were subsequently transferred onto a PVDF membrane (EMD Millipore) and blocked at room temperature with 5% fat-free powdered milk dissolved in tris-buffered saline (TBS) containing 0.1% Tween for 1.5 h. The membrane was incubated with the following primary antibodies at 4̊C overnight: Anti-SIRT1 (1:1,000; Cell Signaling Technology, Inc.), anti-cleaved caspase-3 (1:1,000; Santa Cruz Biotechnology, Inc.), anti-pro-caspase-3 (1:1,000; Santa Cruz Biotechnology, Inc.), anti-GAPDH (1:5,000; Cell Signaling Technology, Inc.), anti-p65 (1:1,000; Abcam) and anti-phosphorylated (p)-p65 (1:1,000; Abcam). Following the primary antibody incubation, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (cat no. ab7090; 1:2,000; Abcam) at room temperature for 2 h. The protein bands were visualized using an enhanced chemiluminescence reagent (EMD Millipore) and GAPDH served as a loading control for normalization.

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Total protein was quantified using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.) and 30 µg protein/lane was separated via 15% SDS-PAGE. The separated proteins were subsequently transferred onto a PVDF membrane (EMD Millipore) and blocked at room temperature using 5% fat-free powdered milk dissolved in TBS-0.1% Tween for 1.5 h. The membranes were incubated with the following primary antibodies at 4˚C overnight: Anti-TLR4 antibody (cat. no. ab13556; 1:1,000; Abcam), anti-GAPDH (cat. no. ab181602; 1:1,000; Abcam), anti-aggrecan (cat. no. ab3778; 1:1,000; Abcam), anti-collagen type II (cat. no. ab34712; 1:1,000; Abcam), anti-p65 (cat. no. ab16502; 1:1,000; Abcam) and anti-phosphorylated (p)-p65 (cat. no. ab86299; 1:1,000; Abcam). Following the primary antibody incubation, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. ab7090; 1:2,000; Abcam) at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence substrate (EMD Millipore) and analyzed using ImageJ version 2.0 software (National Institutes of Health). The expression levels were normalized to GAPDH.