Journal:

Article Title: Base Excision Repair Is Limited by Different Proteins in Male Germ Cell Nuclear Extracts Prepared from Young and Old Mice

doi: 10.1128/MCB.22.7.2410-2418.2002

Figure Lengend Snippet: Western blot analysis of BER proteins in nuclear extracts prepared from enriched populations of primitive type A spermatogonia (PA), type A spermatogonia (A), type B spermatogonia (B), pachytene spermatocytes (P), and round spermatids (R). Bands corresponding to DNA ligase I (130 kDa), DNA ligase III (93 kDa), Xrcc-1 (69 kDa), β-pol (39 kDa), and Ape/Ref-1 (37 kDa) proteins were visualized. Triplicate assays for each of three independent nuclear extract preparations were performed. Some variation between replicates was noted. A summary of all data is shown in Table ​Table1.1. Molecular mass protein standards and purified DNA ligases I and III, β-pol, and Ape/Ref-1 (lane STD) are shown for comparison.

Article Snippet: Rabbit anti-human APE/REF-1 (hAPE/REF-1; Novus Biologicals, Littleton, Colo.) and rabbit polyclonal anti-β-pol (S. Wilson, NIEHS, Research Triangle Park, N.C.) were used to detect Ape/Ref-1 and β-pol, respectively.

Techniques: Western Blot, Purification, Comparison

Journal:

Article Title: Base Excision Repair Is Limited by Different Proteins in Male Germ Cell Nuclear Extracts Prepared from Young and Old Mice

doi: 10.1128/MCB.22.7.2410-2418.2002

Figure Lengend Snippet: Western blot analysis of BER proteins in MGC nuclear extracts prepared from 3-month-old, 16-month-old, and 28-month-old mice. Bands corresponding to DNA ligase I (130 kDa), DNA ligase III (93 kDa), Xrcc-1 (69 kDa), β-pol (39 kDa), and Ape/Ref-1 (37 kDa) proteins were visualized. Triplicate assays were performed for each of three independent nuclear extract preparations. Some variation in signal intensities was observed among Western blots. A summary of all data is shown in Table ​Table2.2. Molecular mass protein standards and purified DNA ligases I and III, β-pol, and APE/REF-1 (lane STD) are shown for comparison.

Article Snippet: Rabbit anti-human APE/REF-1 (hAPE/REF-1; Novus Biologicals, Littleton, Colo.) and rabbit polyclonal anti-β-pol (S. Wilson, NIEHS, Research Triangle Park, N.C.) were used to detect Ape/Ref-1 and β-pol, respectively.

Techniques: Western Blot, Purification, Comparison

Journal:

Article Title: Base Excision Repair Is Limited by Different Proteins in Male Germ Cell Nuclear Extracts Prepared from Young and Old Mice

doi: 10.1128/MCB.22.7.2410-2418.2002

Figure Lengend Snippet: UDG-BER activities for MGC nuclear extracts prepared from 3-month-old B6D2F1 (open), 28-month-old B6D2F1 (solid), and 3-month-old Apex heterozygous knockout mice (striped). Results are presented as means ± SEM of three replicate assays for each of three independent nuclear extract preparations. ∗, significantly (P < 0.05) different from addition of 0 ng of protein within a specific group. †, amount of purified APE/REF-1 required to restore activity to that of 3-month-old mice with 0 ng of protein added.

Article Snippet: Rabbit anti-human APE/REF-1 (hAPE/REF-1; Novus Biologicals, Littleton, Colo.) and rabbit polyclonal anti-β-pol (S. Wilson, NIEHS, Research Triangle Park, N.C.) were used to detect Ape/Ref-1 and β-pol, respectively.

Techniques: Knock-Out, Purification, Activity Assay

Journal: Cancer biology & therapy

Article Title: The combination of 5-fluorouracil plus p53 pathway restoration is associated with depletion of p53-deficient or mutant p53-expressing putative colon cancer stem cells.

doi: 10.4161/cbt.8.22.10446

Figure Lengend Snippet: Figure 1. Upregulation of p53-dependent transcriptional activity or endogenous pro-apoptotic TRAIL receptor DR5 expression following p53 pathway restoration. In the upper panels, a whole population of Phoenix cells expressing a p53-responsive EGFP reporter show increased EGFP reporter expression following infection by a p73-expressing Adenovirus (upper right vs. upper left). In the lower panels, mutant p53-expressing human colon cancer DLD1 cells were treated with 15 μg/ml of the mutant p53 conformation modifying drug CP-31398 for 12 hours followed by fluorescence microscopy for p53 target TRAIL receptor DR5 expression by immunofluorescence with a DAPI nuclear counterstain. The lower left panel shows DR5 expression in untreated DLD1 cells and the lower right panel shows DR5 expression in CP-31398 treated DLD1 cells.

Article Snippet: A rabbit anti-human DR5 IgG antibody (Imgenex) was added at a 1:100 dilution and this was incubated overnight with gentle shaking at 4°C.

Techniques: Activity Assay, Expressing, Infection, Mutagenesis, Fluorescence, Microscopy, Immunofluorescence

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