Prestained lysozyme on the immunoblots is designated as 14.three kD, lysozyme’s reported molecular mass, without regard to changes within the mobility with the marker as a consequence of prestaining. Silver staining of proteins was performed in accordance with the process of Blum (24). The markers employed for silver-stained gels and for the determinations of apparent molecular weights have been the Mark 12 wide-range proteins standards from Novel Experimental Technologies (San Diego, CA).Analysis of HuMig Production by THP-1 Cells and by Peripheral Blood Monocytes. THP-1 cells, derived from a human histiocyticlymphoma, had been obtained from the American Variety Culture Collection, Rockville, MD. Human peripheral blood monocytes had been collected from standard donors by elutriation by the Division of Transfusion Medicine, Clinical Center, National Institutes of Overall health. 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 no or with two,000 U/ml IFN- /. For evaluation of HuMig production by peripheral blood monocytes, cells were incubated at a density of 106 cells/m/for 48 h without having or with 2,000 U/rnl IFN-7. Protease inhibitors leupeptin two p-g/ml, EDTA 1 raM, PMSF 0.five mM, aprotinin two 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) were added to the cell supernatants before the samples had been concentrated for evaluation. Anti-HuMig serum 5092 and protein A-Sepharose (pharmacia LKB Biotechnology, Piscataway, NJ) had been utilised 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 were collected as described above and produced 50 mM Tris/HC1 pH 7.five and 0.five mM EDTA. 8-10 liters of supernatant had been loaded on a carboxymethyl (CM)-cellulose column (TrkB Agonist web MetaChem Technologies Inc., Torrance, CA) mounted on a ConSep LC100 liquid chromatograph (Millipore Co., Milford, MA) as well as the bound proteins have been eluted having 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 had been identified by immunoblotting and peak fractions were pooled, concentrated using the Centriprep-3 device (Amicon Inc., Nav1.7 Antagonist site Beverly, MA), and subjected to reversed phase HPLC on a 46 cm 15 cm C18 column (Vydac, Hesperia, CA). The rHuMig species have been eluted making use of a gradient of 15-40 acetonitrile in 0.05 TFA more than 60 min at a flow price 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 working with a spiral wound CH2 apparatus (Amicon Inc., Beverly, MA), plus the concentrate was dialyzed 1303 Liao et al.against 25 mM Tris/HC1 pH 7.five, 0.five mM EDTA, and 25 NaC1. The dialyzed sample was reapplied towards the CM-cellulose column and also the bound proteins have been eluted with a linear gradient of 0.025-1 M NaC1 in 25 mM Tris/HC1, pH 7.5, 0.five mM EDTA. The ability with the low-kD rHuMig to bind to the CMcellulose column afte.