Prestained lysozyme on the immunoblots is designated as 14.three kD, lysozyme’s reported molecular mass, without regard to alterations inside the mobility from the marker as a result of prestaining. Silver staining of proteins was performed based on the system of Blum (24). The markers Glycoprotein 130 (gp130) Proteins Biological Activity applied for silver-stained gels and for the Decoy Receptor 3 Proteins Accession determinations of apparent molecular weights have been the Mark 12 wide-range proteins requirements from Novel Experimental Technologies (San Diego, CA).Evaluation of HuMig Production by THP-1 Cells and by Peripheral Blood Monocytes. THP-1 cells, derived from a human histiocyticlymphoma, had been obtained in the American Type Culture Collection, Rockville, MD. Human peripheral blood monocytes had been collected from regular donors by elutriation by the Division of Transfusion Medicine, Clinical Center, National Institutes of Wellness. THP-1 cells and monocytes have been cultured routinely in 1KPMI 1640 with ten FCS. For analysis of HuMig production by the THP-1 cell line, cells had been incubated at a density of 106 cells/ml for 30 h with out or with two,000 U/ml IFN- /. For evaluation of HuMig production by peripheral blood monocytes, cells had been incubated at a density of 106 cells/m/for 48 h without or with two,000 U/rnl IFN-7. Protease inhibitors leupeptin two p-g/ml, EDTA 1 raM, PMSF 0.five mM, aprotinin 2 p-g/ml, bestatin ten p-g/ml, calpain inhibitor 17 g/ml, E-64 1 p-g/m/, and pepstatin 0.7 p,g/m/ (Boehringer Mannheim Corp., Indianapolis, IN) have been added towards the cell supernatants prior to the samples have been concentrated for analysis. Anti-HuMig serum 5092 and protein A-Sepharose (pharmacia LKB Biotechnology, Piscataway, NJ) had been applied for iimnunoprecipitation. The precipitates had been analyzed by Tricine-SDS-PAGE and immunoblotting as described above. Purification ofrHuMig Proteins. (a) Purification ofbigh-kD proteins. The culture supernatants from rHuMig-overexpressing C H O / H 9 cells have been collected as described above and produced 50 mM Tris/HC1 pH 7.five and 0.5 mM EDTA. 8-10 liters of supernatant have been loaded on a carboxymethyl (CM)-cellulose column (MetaChem Technologies Inc., Torrance, CA) mounted on a ConSep LC100 liquid chromatograph (Millipore Co., Milford, MA) along with the bound proteins had been eluted using a linear gradient of 0.025-1 M NaC1 in 50 mM Tris/HC1 pH 7.five, 0.5 mM EDTA. The high-kD rHuMig species eluted as a single asymmetrical peak at ,” 0.five M NaC1. Fractions containing rHuMig were identified by immunoblotting and peak fractions had been pooled, concentrated utilizing the Centriprep-3 device (Amicon Inc., Beverly, MA), and subjected to reversed phase HPLC on a 46 cm 15 cm C18 column (Vydac, Hesperia, CA). The rHuMig species had been eluted employing a gradient of 15-40 acetonitrile in 0.05 TFA more than 60 min at a flow rate of I ml/min on a liquid chromatograph (model 1050; Hewlett-Packard Co., Palo Alto, CA). The column eluate was monitored at 280 nm, 205 nm, and, for reference, at 450 nm. The high-kD rHuMig species eluted at ,’- 44 rain. (b) Purification of low-kD species. The flow-through from the CM-cellulose column as described above was collected, concentrated 20-40-fold making use of a spiral wound CH2 apparatus (Amicon Inc., Beverly, MA), along with the concentrate was dialyzed 1303 Liao et al.against 25 mM Tris/HC1 pH 7.five, 0.5 mM EDTA, and 25 NaC1. The dialyzed sample was reapplied towards the CM-cellulose column plus the bound proteins were eluted having a linear gradient of 0.025-1 M NaC1 in 25 mM Tris/HC1, pH 7.5, 0.5 mM EDTA. The potential of the low-kD rHuMig to bind to the CMcellulose column afte.