Journal: Blood

Article Title: Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation

doi: 10.1182/blood-2008-04-151639

Figure Lengend Snippet: Complete blood counts and erythrocyte indices of Atg7 −/− transplant recipients

Article Snippet: 15 , 28 The following primary antibodies were used: 1 μg/mL LC3, mouse immunoglobulin G1 (IgG1) monoclonal antibody; 1 μg/mL GABARAP, mouse IgG1 monoclonal antibody; 1:1000 GATE-16, rabbit polyclonal antibody (Medical and Biological Laboratories); 1:1000 Atg12, rabbit polyclonal antibody (Cell Signaling Technology); 1 μg/mL Atg7, rabbit polyclonal antibody (ProSci); 0.5 μg/mL NIX, rabbit polyclonal antibody (Exalpha Biologicals); 0.1 μg/mL Beclin-1, rabbit polyclonal antibody (Santa Cruz Biotechnology); 0.25 μg/mL Ter119, rat monoclonal antibody (BD Biosciences); and 1:10 000 β-actin, mouse IgG1 monoclonal antibody (Sigma-Aldrich).

Techniques:

Journal: Blood

Article Title: Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation

doi: 10.1182/blood-2008-04-151639

Figure Lengend Snippet: Mitochondrial clearance is preserved in unstressed Atg7−/− transplant recipients. (A) Blood smears from Atg7+/+ and Atg7−/− transplant recipients, stained with Wright-Giemsa (original magnification, ×1000). Note the increased central pallor of the Atg7−/− erythrocytes. (B) Flow cytometry of blood stained with MTR (top panels) or doubly stained with MTR and TO (bottom panels) from Atg7+/+ and Atg7−/− transplant recipients and Nix−/− mice.

Article Snippet: 15 , 28 The following primary antibodies were used: 1 μg/mL LC3, mouse immunoglobulin G1 (IgG1) monoclonal antibody; 1 μg/mL GABARAP, mouse IgG1 monoclonal antibody; 1:1000 GATE-16, rabbit polyclonal antibody (Medical and Biological Laboratories); 1:1000 Atg12, rabbit polyclonal antibody (Cell Signaling Technology); 1 μg/mL Atg7, rabbit polyclonal antibody (ProSci); 0.5 μg/mL NIX, rabbit polyclonal antibody (Exalpha Biologicals); 0.1 μg/mL Beclin-1, rabbit polyclonal antibody (Santa Cruz Biotechnology); 0.25 μg/mL Ter119, rat monoclonal antibody (BD Biosciences); and 1:10 000 β-actin, mouse IgG1 monoclonal antibody (Sigma-Aldrich).

Techniques: Staining, Flow Cytometry

Journal: Blood

Article Title: Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation

doi: 10.1182/blood-2008-04-151639

Figure Lengend Snippet: Mitochondrial clearance is impaired in Atg7−/− reticulocytes. (A) Immunofluorescence and flow cytometry of reticulocyte-enriched blood cultured for 3 days in vitro from wild-type and Nix−/− mice and Atg7−/− transplant recipients. The cells are stained with MTR. The black line in the histograms corresponds to day 0, and the shaded area to day 3. The scale is indicated at the bottom. (B) Flow cytometry of blood from phlebotomized mice stained with MTR (top panels) or doubly stained with MTR and TO (bottom panels). Mice were phlebotomized for 4 days. Thereafter, a small amount of blood (10 μL) was withdrawn on days 0, 1, and 3 for analysis. Day 0 was the day after the final day of phlebotomy. Genotypes are on the left.

Article Snippet: 15 , 28 The following primary antibodies were used: 1 μg/mL LC3, mouse immunoglobulin G1 (IgG1) monoclonal antibody; 1 μg/mL GABARAP, mouse IgG1 monoclonal antibody; 1:1000 GATE-16, rabbit polyclonal antibody (Medical and Biological Laboratories); 1:1000 Atg12, rabbit polyclonal antibody (Cell Signaling Technology); 1 μg/mL Atg7, rabbit polyclonal antibody (ProSci); 0.5 μg/mL NIX, rabbit polyclonal antibody (Exalpha Biologicals); 0.1 μg/mL Beclin-1, rabbit polyclonal antibody (Santa Cruz Biotechnology); 0.25 μg/mL Ter119, rat monoclonal antibody (BD Biosciences); and 1:10 000 β-actin, mouse IgG1 monoclonal antibody (Sigma-Aldrich).

Techniques: Immunofluorescence, Flow Cytometry, Cell Culture, In Vitro, Staining

Journal: Blood

Article Title: Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation

doi: 10.1182/blood-2008-04-151639

Figure Lengend Snippet: Deficient ubiquitin-like conjugation activity in Atg7−/− erythroid cells. (A) Western blot analyses of LC3, GABARAP (GRP), GATE-16 (GT16), Atg12, Ter119, BCL-XL, Beclin-1, and β-actin in expanded E14.5 fetal liver cells undergoing differentiation in culture for 2 days. The upper LC3 band corresponds to the unconjugated form of LC3, and the lower band corresponds to the lipid conjugated form of LC3. This same is true for the other Atg8 homologs, GABARAP and GATE-16. Wild-type murine embryonic fibroblasts (MEF) provide a positive control for the position of the unconjugated and conjugated proteins. Genotypes and days in culture are indicated at the top. (B) Western blot analyses of Atg7, NIX, Beclin-1, and β-actin in primary E14.5 fetal liver cells. NIX protein is expressed as 2 isoforms in erythroid cells, a full-length monomeric form (top band), and a shorter isoform, which has not been fully characterized (bottom band). Beclin-1 is provided as a loading control. Genotypes are indicated at the top.

Article Snippet: 15 , 28 The following primary antibodies were used: 1 μg/mL LC3, mouse immunoglobulin G1 (IgG1) monoclonal antibody; 1 μg/mL GABARAP, mouse IgG1 monoclonal antibody; 1:1000 GATE-16, rabbit polyclonal antibody (Medical and Biological Laboratories); 1:1000 Atg12, rabbit polyclonal antibody (Cell Signaling Technology); 1 μg/mL Atg7, rabbit polyclonal antibody (ProSci); 0.5 μg/mL NIX, rabbit polyclonal antibody (Exalpha Biologicals); 0.1 μg/mL Beclin-1, rabbit polyclonal antibody (Santa Cruz Biotechnology); 0.25 μg/mL Ter119, rat monoclonal antibody (BD Biosciences); and 1:10 000 β-actin, mouse IgG1 monoclonal antibody (Sigma-Aldrich).

Techniques: Ubiquitin Proteomics, Conjugation Assay, Activity Assay, Western Blot, Positive Control, Control

Journal: Blood

Article Title: Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation

doi: 10.1182/blood-2008-04-151639

Figure Lengend Snippet: Atg7-independent mitochondrial clearance is mediated by degradative vacuoles. (Top) Ultrastructure of reticulocytes from Atg7+/+ and Atg7−/− transplant recipients cultured for 1 day. The scale is indicated at the bottom. (Bottom) Quantification of mitochondria and degradative vacuoles in cultured Atg7+/+, Atg7−/−, and Nix−/− reticulocytes expressed as a ratio. Fifty digital images containing 100 to 200 mitochondria and vacuoles per sample were quantified. Genotypes and days in culture are indicated at the bottom.

Article Snippet: 15 , 28 The following primary antibodies were used: 1 μg/mL LC3, mouse immunoglobulin G1 (IgG1) monoclonal antibody; 1 μg/mL GABARAP, mouse IgG1 monoclonal antibody; 1:1000 GATE-16, rabbit polyclonal antibody (Medical and Biological Laboratories); 1:1000 Atg12, rabbit polyclonal antibody (Cell Signaling Technology); 1 μg/mL Atg7, rabbit polyclonal antibody (ProSci); 0.5 μg/mL NIX, rabbit polyclonal antibody (Exalpha Biologicals); 0.1 μg/mL Beclin-1, rabbit polyclonal antibody (Santa Cruz Biotechnology); 0.25 μg/mL Ter119, rat monoclonal antibody (BD Biosciences); and 1:10 000 β-actin, mouse IgG1 monoclonal antibody (Sigma-Aldrich).

Techniques: Cell Culture

Journal: Blood

Article Title: Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation

doi: 10.1182/blood-2008-04-151639

Figure Lengend Snippet: ABT-737 and NIX differ in their requirement for BAX or BAK. (Left panels) Flow cytometry of reticulocyte-enriched blood from phlebotomized Bax−/−;Bak−/−, Atg7+/+, Atg7−/−, and Nix−/− mice cultured for 3 days and doubly stained with MTR and TO. Genotypes are indicated on the left. (Right panels) Flow cytometry of the same blood samples, cultured for 1 day in the presence (shaded area) or absence (black line) of ABT-737, and stained with MTR.

Article Snippet: 15 , 28 The following primary antibodies were used: 1 μg/mL LC3, mouse immunoglobulin G1 (IgG1) monoclonal antibody; 1 μg/mL GABARAP, mouse IgG1 monoclonal antibody; 1:1000 GATE-16, rabbit polyclonal antibody (Medical and Biological Laboratories); 1:1000 Atg12, rabbit polyclonal antibody (Cell Signaling Technology); 1 μg/mL Atg7, rabbit polyclonal antibody (ProSci); 0.5 μg/mL NIX, rabbit polyclonal antibody (Exalpha Biologicals); 0.1 μg/mL Beclin-1, rabbit polyclonal antibody (Santa Cruz Biotechnology); 0.25 μg/mL Ter119, rat monoclonal antibody (BD Biosciences); and 1:10 000 β-actin, mouse IgG1 monoclonal antibody (Sigma-Aldrich).

Techniques: Flow Cytometry, Cell Culture, Staining

Journal: Blood

Article Title: Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation

doi: 10.1182/blood-2008-04-151639

Figure Lengend Snippet: Mitochondrial depolarization is a consequence of autophagosome formation in reticulocytes. Immunofluorescence of reticulocyte-enriched blood from phlebotomized Atg7+/+ and Atg7−/− transplant recipients, cultured for 3 days, and doubly stained with MTG and TMRM. The mean immunofluorescence intensity per cell of all cells with mitochondria in a representative field was quantified in the red and green channels, and the ratio of the signals was determined. The higher the ratio, the greater the average mitochondrial polarization per cell. The proportion of cells within each range is represented by a chart below each picture (legend at the bottom). Approximately 50 to 100 cells were quantified per sample (field). Note that mitochondria in the Atg7−/− reticulocytes remain polarized, whereas mitochondria in Atg7+/+ reticulocytes do not. No quantification is provided for Atg7+/+ reticulocytes on day 3 due to an insufficient number of cells with mitochondria for analysis. The scale is indicated at the bottom.

Article Snippet: 15 , 28 The following primary antibodies were used: 1 μg/mL LC3, mouse immunoglobulin G1 (IgG1) monoclonal antibody; 1 μg/mL GABARAP, mouse IgG1 monoclonal antibody; 1:1000 GATE-16, rabbit polyclonal antibody (Medical and Biological Laboratories); 1:1000 Atg12, rabbit polyclonal antibody (Cell Signaling Technology); 1 μg/mL Atg7, rabbit polyclonal antibody (ProSci); 0.5 μg/mL NIX, rabbit polyclonal antibody (Exalpha Biologicals); 0.1 μg/mL Beclin-1, rabbit polyclonal antibody (Santa Cruz Biotechnology); 0.25 μg/mL Ter119, rat monoclonal antibody (BD Biosciences); and 1:10 000 β-actin, mouse IgG1 monoclonal antibody (Sigma-Aldrich).

Techniques: Immunofluorescence, Cell Culture, Staining

Journal:

Article Title: A comparison of 13 guinea pig and human anti-tissue transglutaminase antibody ELISA kits

doi:

Figure Lengend Snippet: IgA anti-tissue transglutaminase (tTG) antibody values of the patients with coeliac disease (CD), and the non-CD (SBX) and inflammatory bowel disease (IBD) controls in the six human tTG based enzyme linked immunosorbent assay (ELISA) kits, with corresponding receiver operating characteristic (ROC) curves and area under curve (AUC) estimations. The solid lines represent the manufacturers' recommended cut off values and the broken lines represent the ROC plot analysis derived decision thresholds. (A) AESKULISA (Aesku.Lab), (B) The Binding Site, (C) Eurospital, (D) QUANTA Lite (Inova), (E) Orgentec, (F) Varelisa (Pharmacia & Upjohn).

Article Snippet: The solid lines represent the manufacturers' recommended cut off values and the broken lines represent the ROC plot analysis derived decision thresholds. (A) AESKULISA (Aesku.Lab), (B) The Binding Site, (C) Eurospital, (D) QUANTA Lite (Inova), (E) Orgentec, (F) Varelisa (Pharmacia & Upjohn). table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Manufacturer's cut off point ROC plot analysis Assay type/Manufacturer Cut off CD (sensitivity) Non-CD controls (specificity) IBD controls Threshold CD (sensitivity) Non-CD controls (specificity) IBD controls IgA EMA IIF/The Binding Site 1/4 49/49 (100%) 0/34 (100%) 0/30 NA NA NA NA Guinea pig liver tTG based ELISA/The Binding Site 4 U/ml 43/49 (88%) 3/34 (91%) 0/30 3.5 U/ml 43/49 (88%) 3/34 (91%) 0/30 Guinea pig liver tTG based ELISA/Eurospital 5 AU 48/49 (98%) 22/34 (35%) 7/30 9 AU 46/49 (94%) 4/34 (88%) 0/30 Guinea pig liver tTG based ELISA/Genesis Diagnostics 10 U/ml 47/49 (96%) 8/34 (76%) 2/30 15.3 U/ml 44/49 (90%) 4/34 (88%) 1/30 Guinea pig liver tTG based ELISA/ImmuLisa 20 EU/ml 45/49 (92%) 8/34 (76%) 0/30 22.2 EU/ml 44/49 (90%) 6/34 (82%) 0/30 Guinea pig liver tTG based ELISA/ Immunopharmacology Research Diagnostics 25 AU 49/49 (100%) 30/34 (12%) 16/30 76.4 AU 43/49 (88%) 4/34 (88%) 2/30 Guinea pig liver tTG based ELISA/QUANTA Lite 20 units/ml 42/49 (86%) 0/34 (100%) 0/30 14.1 units/ml 45/49 (92%) 1/34 (97%) 1/30 Guinea pig liver tTG based ELISA/Medizyme 25 U/ml 48/49 (98%) 16/34 (53%) 15/30 38.5 U/ml 43/49 (88%) 4/34 (88%) 2/30 Open in a separate window Results equal to or greater than the cut off/threshold were considered positive.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Binding Assay

Journal:

Article Title: A comparison of 13 guinea pig and human anti-tissue transglutaminase antibody ELISA kits

doi:

Figure Lengend Snippet: IgA anti-tissue transglutaminase (tTG) antibody values of the patients with coeliac disease (CD), and the non-CD (SBX) and inflammatory bowel disease (IBD) controls in the six human tTG based enzyme linked immunosorbent assay (ELISA) kits, with corresponding receiver operating characteristic (ROC) curves and area under curve (AUC) estimations. The solid lines represent the manufacturers' recommended cut off values and the broken lines represent the ROC plot analysis derived decision thresholds. (A) AESKULISA (Aesku.Lab), (B) The Binding Site, (C) Eurospital, (D) QUANTA Lite (Inova), (E) Orgentec, (F) Varelisa (Pharmacia & Upjohn).

Article Snippet: The solid lines represent the manufacturers' recommended cut off values and the broken lines represent the ROC plot analysis derived decision thresholds. (A) AESKULISA (Aesku.Lab), (B) The Binding Site, (C) Eurospital, (D) QUANTA Lite (Inova), (E) Orgentec, (F) Varelisa (Pharmacia & Upjohn). table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Manufacturer's cut off point ROC plot analysis Assay type/Manufacturer Cut off CD (sensitivity) Non-CD controls (specificity) IBD controls Threshold CD (sensitivity) Non-CD controls (specificity) IBD controls IgA EMA IIF/The Binding Site 1/4 49/49 (100%) 0/34 (100%) 0/30 NA NA NA NA Guinea pig liver tTG based ELISA/The Binding Site 4 U/ml 43/49 (88%) 3/34 (91%) 0/30 3.5 U/ml 43/49 (88%) 3/34 (91%) 0/30 Guinea pig liver tTG based ELISA/Eurospital 5 AU 48/49 (98%) 22/34 (35%) 7/30 9 AU 46/49 (94%) 4/34 (88%) 0/30 Guinea pig liver tTG based ELISA/Genesis Diagnostics 10 U/ml 47/49 (96%) 8/34 (76%) 2/30 15.3 U/ml 44/49 (90%) 4/34 (88%) 1/30 Guinea pig liver tTG based ELISA/ImmuLisa 20 EU/ml 45/49 (92%) 8/34 (76%) 0/30 22.2 EU/ml 44/49 (90%) 6/34 (82%) 0/30 Guinea pig liver tTG based ELISA/ Immunopharmacology Research Diagnostics 25 AU 49/49 (100%) 30/34 (12%) 16/30 76.4 AU 43/49 (88%) 4/34 (88%) 2/30 Guinea pig liver tTG based ELISA/QUANTA Lite 20 units/ml 42/49 (86%) 0/34 (100%) 0/30 14.1 units/ml 45/49 (92%) 1/34 (97%) 1/30 Guinea pig liver tTG based ELISA/Medizyme 25 U/ml 48/49 (98%) 16/34 (53%) 15/30 38.5 U/ml 43/49 (88%) 4/34 (88%) 2/30 Open in a separate window Results equal to or greater than the cut off/threshold were considered positive.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Binding Assay