Piercetm 660 nm protein assay reagent

The cells harvested from the cultures (see above) were lysed in RIPA buffer for protein extraction. The protein concentration of the extracts was quantified using Pierce^TM 660 nm protein assay reagent (Thermo Fisher Scientific). The extracts (25 μg of total protein per sample) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) as described elsewhere.^{50 (link)} Rabbit polyclonal antibodies raised against HIF-1α (Cayman Chemical, Ann Arbor, MI, USA; dilution 1:200), total NF-κB p65, and phosphorylated NF-κB p65, phosphorylated IKK (Cell Signaling Technology, Danvers, MA, USA; 1:1000) were used in blocking buffer (5% bovine serum albumin in Tris-buffered saline). Anti-rabbit IgG conjugated with horseradish peroxidase (HRP; Cell Signaling Technology; 1:2500) was used as a secondary antibody. Beta-actin served as a loading control and was detected by anti-β-actin antibody conjugated with HRP (Abcam; 1:10,000). Target proteins on the antibody-treated membranes were visualised by an enhanced chemiluminescent method using Luminata Forte Western HRP substrate (EMD Millipore).

LDL was isolated from human plasma using KBr-density gradient as previously described (Havel et al., 1955 (link)). Briefly, plasma was isolated from blood by centrifugation at 2,000 × g, 4°C for 10 min. Plasma density was adjusted to 1.100 g/ml using specific gravity KBr Solution A (density at 1.006 g/ml) and centrifuged at 17,136 × g, 4°C for 18 h using a 70.1 Ti fixed-angle titanium rotor (Beckman Coulter, 342184, Brea, CA) and Optma LE-8OK Ultracentrifuge (Beckman Coulter, 8043-30-1192). The supernatant fraction containing very-low-density lipoprotein (VLDL) was discarded. Solution density was adjusted using specific gravity KBr Solution B (density at 1,182 g/ml) and centrifuged at 17,136 × g, 4°C for 24 h. The supernatant fraction (orange ring) containing low-density lipoprotein (LDL) was isolated and further dialyzed in 0.85% (w/v) NaCl at 4°C for 24 h. LDL was incubated with 10 μM CuSO₄ at 37°C for 24 h. Oxidized LDL (ox-LDL) was dialyzed in 0.85% (w/v) NaCl at 4°C for 24 h. Protein content was determined using Pierce^TM 660 nm Protein Assay reagent (Thermo Fisher Scientific, 22660) and Pierce^TM Bovine Serum Albumin Standard Pre-diluted Set (Thermo Fisher Scientific, 23208) as standards.