Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against .alpha.-transforming growth factor

Abstract

Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human alpha-transforming growth factor (alpha-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting alpha-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native alpha-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of alpha-TGF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of alpha-TGF has a cellular role beyond that of an autocrine growth factor.

Human A673 cells secrete high molecular weight EGF‐receptor binding growth factors that appear to be immunologically unrelated to EGF or TGF‐α

“…activity in conditioned medium of human melanoma cells A-2058 [40]. Synthetic hTGF or synthetic rTGF-a in either its C-terminal (residues 34-50) or full length form (residues 1-50) did react fully [40] (Fig. 5), whereas highly purified hEGF did not react at all.…”

“…Furthermore, as summarized in Table I, neither HPLC peak of AIG activity (added in an amount of 200 ng EGF equivalents by RFL4) was detected by Western immunoblots under conditions in which 50-75 ng of synthetic rTGF-a or hTGF-a! were clearly detected [40].…”

“…Detectability (10% competition) is 0.3 ng synthetic rTGF-a reference standard and in which 50% competition occurs with 0.5 ng competing synthetic rTGF-a or hTGF-a under reduced and denatured conditions. 'rTGF-a immunoblot [40] sensitive to detection of 50-75 ng of synthetic rTGF-a or hTGF-a. dHighly purified hEGF [39].…”

“…Highly purified hEGF [39] was used as '251-labeled tracer and reference standard. TGF-a RIA was performed using a commercial kit (Biotope, Inc., Seattle, WA), and as described [40], with bioactive synthetic rTGF-a (Peninsula Laboratories, Belmont, CA) used as radioiodinated tracer and reference standard. Antisera to the C-terminal 17-amino acid fragment (C17A) of rTGF-a were raised either in rabbits (Biotope, Inc.) or as an affinity-purified antiserum raised in sheep [40].…”

“…All TGF-a RIAs using either rabbit or sheep antisera detected hTGF-a and rTGF-a equally well. Western immunoblots [40] for TGF-a were 78:CMBT A673 TGFs Immunologically Distinct From TGF-a JCB:251 carried out on the two biologically active HPLC components; the technique detected 50-75 ng of either rTGF-a, or hTGF-a, as reference standards. Synthetic hTGF-a, was the gift of Dr. J.E.…”