Total lysates were prepared, and Western analysis was performed
Total lysates were prepared, and Western analysis was performed. and and LDL receptorCdeficient (and and mice or from transgenically rescued IR KO mice and littermate mice (19, 20) were collected by peritoneal lavage, and pooled macrophages of each strain were cultured as explained (21), except that 0.2% bovine serum albumin (BSA) was supplemented in DMEM (Invitrogen, Carlsbad, California, USA) containing 1,000 mg/l of D-glucose unless otherwise specified. After 1C2 hours, the cells were washed three times with 1 phosphate-buffered saline (PBS; Invitrogen) and were used MK-4101 immediately for experiments as explained below. For the isolation of mouse peripheral blood monocytes, the flotation method (22) with OptiPrep density gradient medium (Sigma-Aldrich, St. Louis, Missouri, USA) was used. Blood from and mice was collected and mixed well with OptiPrep in Tricine-buffered saline (TBS; 0.85% NaCl and 10 mM Tricine-NaOH, pH 7.4). The pooled samples were centrifuged at 1,000 for 30 minutes at room heat. The cells were harvested from your meniscus downwards and diluted into TBS. After being spun at 400 for 10 minutes, the cells were resuspended MK-4101 and cultured as explained above. Acetylated LDL (acLDL) uptake and cholesteryl ester formation. We prepared acLDL and labeled it with 125I and 3H as explained (21, 23). The assays of cell association, degradation, and cholesteryl ester formation with macrophages following MK-4101 loading of [125I,3H]acLDL were performed as previously explained (23, 24). Binding assay for oxLDL. 125I-labeled oxLDL was prepared as explained (25). Labeled oxLDL (10C25 g/ml or in amounts as indicated in Physique ?Physique1E)1E) in DMEM/0.2% BSA with or without the addition of unlabeled oxLDL (40-fold excess; for estimates of nonspecific binding) were incubated for 2C3 hours at 4C with macrophages with or without prior treatments as indicated in physique legends. In some experiments, macrophages were preincubated for 30 minutes at 4C with fucoidan (Sigma-Aldrich), mouse antiCCD36 IgA (Cascade BioScience, Winchester, Massachusetts, USA), or control mouse IgA (Biodesign, Saco, Maine, USA) in DMEM/0.2% BSA prior to the addition of oxLDL. After cells were washed on ice three times with ice-cold PBS, cell-associated 125I radioactivity was decided as explained above. Open in a separate window Physique 1 Enhanced uptake of altered LDL in versus WT mouse peritoneal macrophages is usually mediated by increases in cell surface expression of CD36 and SR-A. (ACC) [125I,3H]acLDL cell association (A), degradation (B), and cholesteryl ester formation (C) are higher in than in WT macrophages following acLDL loading. One representative experiment of three impartial experiments each using pooled macrophages from five WT and seven mice is usually shown. Short-term treatment (5 hours) of macrophages with either insulin or leptin does not switch these parameters. C CE (vertical axis, C), cholesterol to cholesteryl ester. (D) Protein expression of scavenger receptors CD36 and SR-A is usually increased while SR-BI expression is decreased in versus WT macrophages, as determined by Western analysis (left). Protein extracts were prepared from pooled macrophages of five WT and five mice. One experiment representative of IL10B four impartial experiments is shown. CD36 and SR-A mRNA was not upregulated in versus WT macrophages, as shown by Northern analysis (right). Northern analysis was performed with random-primed CD36, SR-A, and actin cDNA probes using total RNA isolated from pooled macrophages of ten mice of each strain. One experiment representative of three impartial experiments is shown. (E) Specific binding of oxLDL to macrophages is usually elevated. (F) Effects of fucoidan and anti-CD36 antibody on oxLDL binding to and WT macrophages. [125I]oxLDL binding assays were MK-4101 performed with pooled macrophages isolated from five mice of each strain, preincubated with the SR-A ligand fucoidan (20 g/ml), mouse antiCCD36 IgA (20 g/ml), or mouse control IgA (20 g/ml, not shown). The decreases in total oxLDL binding in the presence of CD36 IgA or fucoidan were considered as binding mediated by CD36 or SR-A, respectively. CD36 contributes more to the increase in oxLDL binding to versus WT macrophages than SR-A. One experiment representative of three impartial experiments is shown. CD36-depend. and SR-A depend., oxLDL binding mediated by CD36 and SR-A, respectively. Ex lover vivo treatments of macrophages and monocytes. New peritoneal macrophages or blood monocytes were treated at 37C in DMEM/0.2% BSA with insulin (Sigma-Aldrich), wortmannin.