Sr bi

At the end of the incubation with glucose, cells were lysed in M-PER Mammalian Protein Extraction buffer (Pierce, Rockford, IL), and protein concentrations were quantified by Lowry protein assay (Bio-RAD, Hercules, CA). Aliquots of total cell lysates obtained from NHMC or HK-2 cells under control or experimental conditions, or from renal cortical tissue (20 µg total protein as determined by the Lowry protein assay) were denatured with sample loading buffer containing 5% β-mercaptoethanol (Invitrogen, Grand Island, NY). Samples were then heated at 95°C for five minutes and subjected to SDS-PAGE followed by Western blot. Membranes were extensively washed with TBS-T in between all steps. Membranes were probed respectively with primary antibodies of ABCA1 (Novus Biologicals, St. Louis, MO), ABCG1 (Santa Cruz Biotechnology, Dallas, TX), SR-BI (Novus Biologicals, St. Louis, MO), CD36, SR-AI, LOX-1, LXR-α, and LXR-β (Abcam, Cambridge, UK) (diluted 1∶1,000), followed by the respective secondary antibodies conjugated to HRP (Cell Signaling Technology, Danvers, MA) (diluted 1∶2,500). Protein bands were visualized with ECL Plus according to the manufacturer's instructions (GE Healthcare, Buckinghamshire, UK).

Mouse anti-ICAM-1(Cat#: sc107), VCAM-1(Cat#: sc13160), GAPDH (Cat#: sc365062) SRA (Cat#: sc56777), αSMA (Cat#: sc130617), CD36 (Cat#: sc70644), and CD68 (Cat#: sc20060) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Rabbit anti-ABCG1 (Cat#: NB400-132), ABCA1(Cat#: NB400-105) and SRBI (Cat#: NB400-104) polyclonal antibodies were purchased from Novus Biologicals (Littleton, CO). Mouse anti-IL-1β (Cat#: #12242) monoclonal antibody was purchased from Cell Signaling Technology. Mouse anti-Arg (Cat#: ab239731) monoclonal antibody were purchased from Abcam (Cambridge, MA). Mouse anti-rabbit IgG-R (Cat#: sc2492), mouse anti-rabbit IgG-FITC (Cat#: sc2359) and m-IgGκ BP-FITC (Cat#: sc516140) antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Astragalus flavone (Cat#: SA9780) was purchased from Solarbio (Beijing, China).

PS (Avanti) was loaded onto nitrocellulose membrane-C (HyBond) (14 (link)) and incubated with glutathione S-transferase (GST)-tagged SR-BI (Novus) or GST alone. The bound protein was detected by anti-SR-BI (Novus) or anti-GST (Novus) and secondary antibody (Sigma). For a cell-based assay, apoptosis was induced in SR-BI^−/− macrophages, and exposure of PS was analyzed by annexin V directly or by incubation with GST-SR-BI protein or GST alone. SR-BI was detected using anti-SR-BI antibody.