Ab90947

Western blotting was performed using a rabbit polyclonal anti-HPX antibody from Abcam (ab90947) and anti-AP2 and anti-GAPDH antibodies from Cell Signaling Technology (D25B3; D16H11). Serum triglyceride and cholesterol levels were measured using reagents from Thermo Scientific (TR13421; TR22421). Serum glucose and free-fatty acids were measured using reagents from Wako Life Sciences (439–90901; 995–34693 and 997–34893). Serum HPX and HPX in cell culture media were measured using an Abcam HPX Elisa kit for mouse (ab157716). Plasma HPX and carotid plaque HPX content were measured using an Abcam HPX Elisa kit for human (ab108859). Carotid plaque total and free cholesterol contents were measured using reagents from Wako Life Sciences (NC9138103; NC9506034). Esterified cholesterol was estimated by subtracting free cholesterol values from total cholesterol values. Carotid plaque analyses were all normalized to protein content of the homogenate. All assays were performed in duplicate with the exception of those on cell culture media, which were performed in triplicate.

Duodenal epithelial scrapings, liver (ground by mortar and pestle), and serum samples (vehicle and 30 μg/kg TCDD groups) were sonicated on ice in RIPA buffer supplemented with protease inhibitor cocktail (Sigma). Samples were centrifuged and total protein in the supernatant was measured using the bicinchoninic acid (BCA) assay (Sigma). The WES capillary electrophoresis system (ProteinSimple, San Jose, CA) was used with the following antibodies: ACO1 (1:65; ab126595; Abcam, Cambridge, MA), FTH1 (1:65; ab65080; Abcam), FTL1 (1:65; ab69090; Abcam), HAMP/HAMP2 (1:10; ab190775; Abcam), HAO1 (1:30; ab93137; Abcam), HP (1:650; ab131236; Abcam), HPX (1:10; ab90947; Abcam), IREB2 (1:65; ab181153; Abcam), SLC11A2 (DMT1; 1:10; ab123085; Abcam), SLC40A1 (1:30; NBP1-21502; Novus Biologicals, Littleton, CO), TFRC (1:30; ab84036; Abcam), and TRF (1:30; ab82411; Abcam). Primary antibodies were detected using a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. Chemiluminescence signals were analyzed with Compass software (ProteinSimple). Protein levels in the duodenum and liver were normalized to SDHA levels, while protein levels in the serum were normalized to sample volume.