Journal: Journal of Biological Chemistry

Article Title: Scavenger Receptor Class B Type I-mediated Reverse Cholesterol Transport Is Inhibited by Advanced Glycation End Products

doi: 10.1074/jbc.m011613200

Figure Lengend Snippet: FIG. 1. Detection of SR-BI protein in CHO-SR-BI cells by im- munoblot analysis. Cell lysates from CHO-SR-BI cells (1st lane, 30 mg), CHO-mock cells (2nd lane, 30 mg), and rat liver parenchymal cells (3rd lane, 30 mg) were subjected to 8% SDS-polyacrylamide gel electro- phoresis and immunoblotted with anti-mouse SR-BI antibody.

Article Snippet: Rabbit polyclonal anti-mouse SR-BI antibody was purchased from Novus Biologicals.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Scavenger Receptor Class B Type I-mediated Reverse Cholesterol Transport Is Inhibited by Advanced Glycation End Products

doi: 10.1074/jbc.m011613200

Figure Lengend Snippet: FIG. 2. Endocytic uptake of 125I-AGE-BSA by CHO-SR-BI cells. Cells were incubated for 5 h in 0.5 ml of 3% BSA in DMEM with increasing concentrations of 125I-AGE-BSA in the presence (closed squares) or absence (closed circle) of 20-fold excess amounts of the unlabeled ligands. After incubation, the medium was taken from each well, and radioactivity soluble in trichloroacetic acid was determined as an index of cellular degradation (B). After incubation, the cells were washed three times with 1 ml of 3% BSA in PBS and then three more times with PBS, lysed with 1 ml of 0.1 N NaOH for 30 min at 37 °C, and the cell-associated radioactivity determined (A). The specific (closed triangles) cell association and degradation were plotted after correcting for nonspecific cell association and degradation. Data represent the means of three separate experiments. Error bars represent S.D.

Article Snippet: Rabbit polyclonal anti-mouse SR-BI antibody was purchased from Novus Biologicals.

Techniques: Incubation, Radioactivity

Journal: Journal of Biological Chemistry

Article Title: Scavenger Receptor Class B Type I-mediated Reverse Cholesterol Transport Is Inhibited by Advanced Glycation End Products

doi: 10.1074/jbc.m011613200

Figure Lengend Snippet: FIG. 3. Binding of 125I-AGE-BSA to CHO-SR-BI cells. Cells were incubated for 90 min at 4 °C in 0.5 ml of 0.1% BSA in DMEM with increasing concentrations of 125I-AGE-BSA in the presence (closed squares) or absence (closed circle) of 20-fold excess amounts of unlabeled ligands. The cells were then washed and lysed in 0.1 N NaOH, and cell-bound radioactivity was determined. Specific binding (closed trian- gles) was determined by subtracting nonspecific binding (closed squares) from total binding (closed circle). Inset, Scatchard analysis of the specific binding curve.

Article Snippet: Rabbit polyclonal anti-mouse SR-BI antibody was purchased from Novus Biologicals.

Techniques: Binding Assay, Incubation, Radioactivity

Journal: Journal of Biological Chemistry

Article Title: Scavenger Receptor Class B Type I-mediated Reverse Cholesterol Transport Is Inhibited by Advanced Glycation End Products

doi: 10.1074/jbc.m011613200

Figure Lengend Snippet: FIG. 4. Effect of modified LDLs and native lipoproteins on cellular bind- ing of 125I-AGE-BSA to CHO-SR-BI cells. A, 125I-AGE-BSA binding. Cells were incubated at 4 °C for 90 min with 0.5 ml of 0.1% BSA in DMEM containing 5 mg/ml 125I-AGE-BSA in the presence or absence of a 20-fold amount of unlabeled AGE-BSA, Ox-LDL, acetyl-LDL, LDL, HDL, or nonglycated BSA. B, 125I-HDL binding. Cells were incubated at 4 °C for 90 min with 0.5 ml of 0.1% BSA in DMEM containing 5 mg/ml 125I-HDL in the pres- ence or absence of a 20-fold amount of unlabeled HDL, AGE-BSA, or non-gly- cated BSA. The amounts of 125I-AGE-BSA bound (A) and 125I-HDL bound (B) were determined as described under “Experi- mental Procedures.” Data represent the means of three separate experiments. Er- ror bars represent S.D.

Article Snippet: Rabbit polyclonal anti-mouse SR-BI antibody was purchased from Novus Biologicals.

Techniques: Modification, Binding Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: Scavenger Receptor Class B Type I-mediated Reverse Cholesterol Transport Is Inhibited by Advanced Glycation End Products

doi: 10.1074/jbc.m011613200

Figure Lengend Snippet: FIG. 5. Effect of modified LDLs and native lipoproteins on endocytic up- take by CHO-SR-BI cells. Cells were incubated at 37 °C for 5 h with 0.5 ml of 3% BSA in DMEM containing 5 mg/ml 125I-AGE-BSA in the presence or absence of a 20-fold amount of unlabeled AGE- BSA, Ox-LDL, acetyl-LDL, LDL, HDL, or non-glycated BSA. The amounts of cell- associated 125I-AGE-BSA (A) and its deg- radation products (B) were determined as described under “Experimental Proce- dures.” Data represent the means of three separate experiments. Error bars repre- sent S.D.

Article Snippet: Rabbit polyclonal anti-mouse SR-BI antibody was purchased from Novus Biologicals.

Techniques: Modification, Incubation

Journal: Journal of Biological Chemistry

Article Title: Scavenger Receptor Class B Type I-mediated Reverse Cholesterol Transport Is Inhibited by Advanced Glycation End Products

doi: 10.1074/jbc.m011613200

Figure Lengend Snippet: FIG. 6. Effect of AGE-BSA on the selective uptake of HDL-CE by CHO-SR-BI cells. CHO-SR-BI cells were incubated for 5 h in 0.5 ml of 3% BSA in DMEM containing 10 mg/ml 125I-HDL-[3H]cholesteryl oleate ether in the presence or absence of increasing concentrations of unlabeled HDL (closed squares), AGE-BSA (closed circle), or non-gly- cated BSA (closed triangles). Under the identical conditions, CHO-mock cells (open circle) were incubated with 10 mg/ml 125I-HDL-[3H]cho- lesteryl oleate ether in the absence of competitors. Selective uptake of HDL-CE was determined as described under “Experimental Proce- dures.” Data represent the means of three separate experiments. Error bars represent S.D.

Article Snippet: Rabbit polyclonal anti-mouse SR-BI antibody was purchased from Novus Biologicals.

Techniques: Incubation

Journal: Journal of Biological Chemistry

Article Title: Scavenger Receptor Class B Type I-mediated Reverse Cholesterol Transport Is Inhibited by Advanced Glycation End Products

doi: 10.1074/jbc.m011613200

Figure Lengend Snippet: FIG. 8. Effect of AGE-BSA on [3H]cholesterol efflux to HDL from CHO-SR-BI cells. A, time course of [3H]cholesterol efflux from CHO-SR-BI. CHO-SR-BI cells were labeled overnight with [3H]cholesterol (1 mCi/ml), incubated for 1 day in 0.5 ml of 0.5% BSA in DMEM, washed, and incubated with HDL (50 mg/ml) (closed circle) in the presence or absence of AGE-BSA (100 mg/ml) (closed triangles) or nonglycated BSA (100 mg/ml) (open squares), or incubated without HDL (open circle). Efflux values are expressed as [3H]cholesterol released to the medium. Data represent the means of three separate experiments. Error bars represent S.D. B, cholesterol efflux to HDL from cells incubated with different concentrations of competitors. CHO-SR-BI and CHO-mock cells were incubated for 5 h with HDL (50 mg/ml) in the presence or absence of different concentrations of AGE-BSA (CHO-SR-BI, closed circle; CHO-mock, open circle) or nonglycated BSA (CHO-SR-BI, closed squares; CHO-mock, open squares). Values are averages of duplicates representative of two separate experiments.

Article Snippet: Rabbit polyclonal anti-mouse SR-BI antibody was purchased from Novus Biologicals.

Techniques: Labeling, Incubation