Biosynthesis of proHNP and HNP in human BM. (A) Myeloblasts/promyelocytes were isolated from human BM by density centrifugation and subjected to a mixed ELISA: immunoplates were incubated with anti-serglycin Ab. Plates were washed, blocked, and incubated with cell lysate. Association of a protein with serglycin was probed with biotinylated Abs against proHNP, which does not recognize fully processed HNP-1, and against HNP-1, which does recognize fully processed HNP-1. After incubation with HRP-conjugated avidin, a color reaction was developed using OPD tablets. Color reaction was measured at an absorbance (Abs) of 492 nm and background reactivity subtracted (absorbance in wells with lysis buffer without cells). Absorbance points to an association of a protein with serglycin. (B) Human BM cells were sedimented with dextran. Supernatant was laid on Lymphoprep and centrifuged at 400g for 30 minutes. Interphase cells were depleted of nongranulocytic cells by immunomagnetic sorting, spun onto slides, and May-Grünwald-Giemsa stained. Bar represents 20 μm. (C) Purified granulocytic precursors were electroporated with siRNA against serglycin, control siRNA, or without siRNA and incubated for 20 hours in a humidified incubator with 5% CO2 at 37°C. Comparative quantification of mRNA for HNP-1 and serglycin. Figure depicts expression levels relative to cells electroporated without siRNA. (D) Transfected granulocyte precursors were pulsed with 35S-methionine/cysteine for 1.5 hours and chased for 3 hours. Cell lysates and medium were immunoprecipitated with Abs against proHNP followed by HNP. Immunoprecipitates were pooled and analyzed by 16% SDS-Tricine-PAGE and fluorography. (E) Ratio of fully processed HNP compared with proHNP in cells and chase medium.
