1. by Véronique Le Cabec, Jero Calafat, and Niels Borregaard
Sorting of the Specific Granule Protein, NGAL, During Granulocytic Maturation of HL-60 Cells
by Véronique Le Cabec, Jero Calafat, and Niels Borregaard
Blood
Volume 89(6):
March 15, 1997
©1997 by American Society of Hematology

2. Biosynthesis of MPO and HLA in undifferentiated and differentiated HL-60 cells.
Biosynthesis of MPO and HLA in undifferentiated and differentiated HL-60 cells. Wild-type (WT) and three clones (A, B, and C) of NGAL-transfected HL-60 cells were pulse-labeled with [35S]methionine for 3 hours, washed, and resuspended at 106 cells/mL in complete medium. Two milliliters was withdrawn for immunoprecipitation with affinity-purified rabbit antibodies against MPO or the β2-microglobulin part of the HLA complex. The fluorograms were developed for 24 hours. The positions of MPO and HLA are indicated with arrows on the right. Molecular weight markers are shown on the left.
Véronique Le Cabec et al. Blood 1997;89:
©1997 by American Society of Hematology

3. Véronique Le Cabec et al. Blood 1997;89:2113-2121
©1997 by American Society of Hematology

4. Véronique Le Cabec et al. Blood 1997;89:2113-2121
©1997 by American Society of Hematology

5. Biosynthesis and sorting of NGAL in undifferentiated and differentiated transfected HL-60 cells after DFP treatment.
Biosynthesis and sorting of NGAL in undifferentiated and differentiated transfected HL-60 cells after DFP treatment. Undifferentiated (A) and differentiated (B) transfected HL-60 cells were pulse-labeled with [35S]methionine as described in the legend of Fig 2, and DFP (final concentration, 25 mmol/L) was added 30 minutes before the end of the pulse. At indicated points, 2 × 106 cells were withdrawn and NGAL was immunoprecipitated from the cell lysates (see legend of Fig 2). The fluorograms were exposed for 48 (A) and 96 hours (B). The results of one experiment representative of two are shown.
Véronique Le Cabec et al. Blood 1997;89:
©1997 by American Society of Hematology

6. Quantification of NGAL and MPO in cells and culture medium by ELISA
Quantification of NGAL and MPO in cells and culture medium by ELISA. NGAL and MPO in undifferentiated (ND) and differentiated (D + RA) cells (▧) and their corresponding media (□) were assayed by ELISA. Release was calculated as the amount of the proteins in...
Quantification of NGAL and MPO in cells and culture medium by ELISA. NGAL and MPO in undifferentiated (ND) and differentiated (D + RA) cells (▧) and their corresponding media (□) were assayed by ELISA. Release was calculated as the amount of the proteins in medium, given as the percentage of the total amount in medium plus cells. Mean ± SD is given. The secretion of MPO from undifferentiated cells was not significantly different from the secretion of MPO from differentiated cells (difference, 0.9% ± 8.8% [n = 18]), whereas secretion of NGAL from differentiated cells was significantly higher than that from undifferentiated cells (difference, 41% ± 8% [n = 13]).
Véronique Le Cabec et al. Blood 1997;89:
©1997 by American Society of Hematology

7. Immunocytochemistry of undifferentiated and differentiated transfected HL-60 cells.
Immunocytochemistry of undifferentiated and differentiated transfected HL-60 cells. Cytospin of undifferentiated (left panels) and DMSO/RA-differentiated (right panels) transfected HL-60 cells containing NGAL cDNA were fixed in 4% formaldehyde in 0.1 mol/L phosphate buffer, permeabilized with Triton X-100, and labeled with affinity-purified rabbit anti-NGAL antibody (2.5 μg/mL; upper panel) or anti-MPO antibody (76 μg/mL; lower panel).
Véronique Le Cabec et al. Blood 1997;89:
©1997 by American Society of Hematology

8. Immunoelectron microscopy of differentiated transfected HL-60 cells.
Immunoelectron microscopy of differentiated transfected HL-60 cells. (A) Labeling of NGAL with rabbit antibody followed by 10 nm gold attached to goat antirabbit IgG. Staining is observed on Golgi stacks (G) and trans-Golgi network (TGN). (B) Labeling of cytochrome b558 with MoAb followed by rabbit antimouse IgG and 10 nm gold attached to goat antirabbit IgG. Extensive labeling can be observed on the plasma membrane (large arrows) and on some vesicles underneath the cell surface (small arrows). No label is observed in the granules (g); nucleus (n). Bars, 200 nm.
Véronique Le Cabec et al. Blood 1997;89:
©1997 by American Society of Hematology