Ab16947

Human hepatocellular carcinoma (HepG2) cells (American Type Culture Collection, Manassas, VA) were maintained in Advanced Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Bio International, Auckland, New Zealand), 2 mM L-glutamine, 0.25 μg/mL amphotericin B, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies) at 37°C in a humidified environment with 5% CO₂. HepG2 cells were seeded at 5×10⁵ cells/mL. 24 hours after seeding, cells were treated with 5 μg/ml of either purified Lp(a) or LDL for 6 hours at 37°C. Cell lysates were harvested and 40 μg of cell lysate protein was subject to western blot analysis with anti-Gpx1 (ab108427, Abcam), anti-Prdx6 (ab16947 Abcam), and anti-SOD (ab13498 Abcam) antibodies. An anti-actin antibody (Novus NB100-74340) was used as a loading control. Protein levels were quantified by densitometry using Image Studio Lite (LI-COR Biosciences, Inc) after normalisation to actin.

Samples of arterial wall and intra-luminal thrombus obtained from AAA patients were embedded in paraffin, and 4 μm cross-sections were cut. Ceroids were detected by direct observation of tissue by fluorescence microscopy (ceroids autofluoresce at 550 nm, producing a red signal). Immunohistochemistry was performed with antibodies against the following proteins: PRDX6 (ab16947, abcam), the lipid peroxidation marker MDA (ab6463, abcam), the neutrophil marker CD15 (Dako), and alpha smooth muscle actin (Dako). Sections were then incubated with the appropriate biotinylated secondary antibody and ABComplex, followed by staining with 3,30-diaminobenzidine (DAB), hematoxylin counterstaining, and mounting in DPX medium.

Equal amounts of HDL (20 μg) were loaded onto 10% polyacrylamide gels, electrophoresed and transferred to nitrocellulose membranes. Blots were blocked with 7% died skimmed milk in 0.05% Tris-buffered saline and Tween (TBS-T) for 1 hour and incubated overnight at 4 °C with the following antibodies: anti-PRDX6 (ab16947, abcam), anti-HLA-I (LS-B6775, LifeSpan Biosciences, Inc), anti-paraoxonase (PON1) (ab24261, abcam), anti-ApoA1 (home-made), or anti-catalase (ab52477, abcam). ApoA1 was detected as a loading control. Membranes were washed with TBS-T and incubated with the appropriate secondary antibody (1:2500) for 1 hour at room temperature. After 4 washes, the signal was detected with an ECL chemiluminiscence kit (GE Healthcare).