Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin

Abstract

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp6Osrc, are described. All Mabs reacted with pp6Ov-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60Ocsrc from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp6O-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp6O-src in a Western immunoblot, and several were used to localize pp6Ov-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy. * Corresponding author. MATERIALS AND METHODS Cells, viruses, and plasmids. Primary cultures of CE cells were prepared from 10-day-old gram-negative, chf-negative, Marek's disease-negative embryos (SPAFAS, Norwich, Conn.) and maintained in culture as described previously (30). Secondary cultures of CE cells were infected with the following strains of RSV: PrA-RSV, SRD-RSV, B77C-RSV, or mutants of PrA-RSV, tsCHdlll9, CHdl121, or CHdl300 (6; J.

Down‐modulation of EGF receptors in cells transformed by the src oncogene

“…Thomas Parsons. A monoclonal antibody to pp6OSrc was obtained from Dr. Sarah Parsons (Parsons et al, 1984).…”

“…Immune complex protein kinase assays were carried out according to Parsons et al (1984). The immune complex was resuspended in buffer containing 20 mM Pipes pH 7.2, 10 mM MnC12, and 10 pCi 32P-ATP and incubated for 30 min at 22°C.…”

pp60c‐src Enhances the Acetylcholine Receptor‐Dependent Catecholamine Release in Vaccinia Virus‐Infected Bovine Adrenal Chromaffin Cells

“…Lysates were centrifuged for 5 min (10,000 g at 4°C). Aliquots containing equal protein were immunoprecipitated with the c-src-specific monoclonal antibodies (mAbs), EClO (which is specific for residues 28-38 of chicken c-src), GDI 1 [which is dependent on residue 1 10 in exogenous (chicken) pp60"" for binding and cross-reacts with endogenous (bovine) pp60c-src; Parsons et al, 1984, 19861, or 2-17 [raised against amino acid residues 2-I7 of src (obtained from Microbiological Associates, Bethesda, MD, U.S.A.)], or with a control antibody, SP2/0, and protein A Sepharose (Sigma) as previously described (Parsons et al, 1984). Immune complexes were washed one time with each of the following buffers: RIPA buffer, HS buffer (50 m M Tris-HC1, pH 7.2, 0.1% NP40, 1 M NaCI, 0.5% aprotinin, 0.0 1 % leupeptin, 1 mM sodium orthovanadate), HO buffer (50 mMTns-HC1, pH 7.2, 1% leupeptin, 1 m M sodium orthovanadate), and HBS (20 mMHEPES, pH 7.2, 150 mM NaCI).…”

Modulation of pp60c‐src tyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells

“…Cells were plated at a density of 2 X 10' per cm2 on collagen-coated cover slips and prepared for immunofluorescent staining with pp60"-"" specific Mabs GDI 1 and EB8, as previously described (Parsons et al, 1984). differentiated tissues also suggests an involvement of pp60'-"' in the specialized functions of these tissues.…”

“…Cells were solubilized in either RIPA buffer (50 mM Tris-C1, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1% Na deoxycholate, 0.1% Na dodecylsulfate, 0.5% aprotinin) or HO buffer (50 mM Tris-HCI, pH 7.2, 1% NP40, 0.5% Na deoxycholate, 100 mM NaCl, 1 mM EDTA, 0.5% aprotinin). Extracts were clarified by centrifugation at 100,OOOg for 30 min, and aliquots adjusted to equal protein content were immunoprecipitated with 1 pl ascites fluid containing either the anti-pp60"-""' Mab, GDI 1, or control antibody, SP2/0, and Pansorbin (Calbiochem, La Jolla, CA) as previously described (Parsons et al, 1979(Parsons et al, , 1984. The derivation, specificity and epitope recognition domain of GDl 1 have been reported elsewhere (Parsons et al, 1984.…”