Biosynthesis and sorting of NGAL in undifferentiated and differentiated transfected HL-60 cells. Undifferentiated (A) and differentiated (B) HL-60 cells transfected with NGAL cDNA under control of a CMV promoter were pulse-labeled with [³⁵S]methionine for 3 hours followed by chase for up to 8 hours. At indicated points, 2 × 10⁶ cells were withdrawn, pelleted by centrifugation, and solubilized. Immunoprecipitation with anti-NGAL antibody was performed on the cell lysates (Cells) and the corresponding culture supernatant (Medium). In addition, NGAL was immunoprecipitated from the supernatant obtained after the 3-hour pulse (S1). The immunoprecipitates (10⁶ cell equivalents) were subjected to a 10% SDS-PAGE and visualized by fluorography. The fluorograms were exposed for 24 hours (A) and 7 days (B). The position of NGAL is indicated with arrows on the right. Molecular weight markers are shown on the left. The results of one experiment, representative of four, are shown. The lower panel of the figure shows the quantification of the radioactivity in NGAL extracted from the gels. Lines are results from cells; hatched bars are from the corresponding culture media; and filled bars are S1, medium from pulse. Results are expressed as the percentage of total labeled NGAL present at the start of chase. Error bars represent the SD of four independent experiments.