A basic phosphoprotein defined by a monoclonal antibody named AF5 was found to be highly abundant in human hepatocellular carcinoma by Western immunoblotting. Under the same conditions, the levels of this phosphoprotein were low or undetectable in normal liver extracts. The AF5 antibody was used to screen a cDNA expression library of a human hepatoma cel line named FOCUS. A 960-base-pair cDNA was isolated and found to be a partial cDNA encoding the human protein-tyrosine kinase substrate p36, also known as lipocortin II. p36 expression was highly abundant in hepatocellular carcinomas at both the transcript and protein levels. Its expression was not induced significantly during rat liver regeneration following a partial hepatectomy. These results suggest that the induction of p36 expression is associated with malignant transformation of hepatocytes. p36 was previously shown to be phosphorylated upon transformation of normal fibroblasts by retroviral oncogenes without significant modulation of expression. We report here the initial description of the association of increased p36 expression with malignant transformation.Several proteins differentially expressed in tumors have been identified by monoclonal antibodies (MAbs) produced against tumor extracts, whole cells, or membrane preparations (for reviews, see references 37 and 38). In order to identify proteins that are present in human hepatocellular carcinoma (HCC) but undetectable in normal liver, we have generated MAbs to a human hepatoma cell line named FOCUS (29, 41). One of these MAbs, named AF5, has allowed us to characterize a basic phosphoprotein antigen. This antigen was not detectable in normal hepatocytes but was present at high levels in primary tumors of hepatocellular origin as well as in HCC-derived cell lines. In this report, we describe the molecular characterization of this phosphoprotein antigen and its identification by cDNA cloning as the protein kinase substrate p36 (21). number of living cells suspended in 0.01 M sodium phosphate (pH 7.2) and 0.14 M sodium chloride buffer (phosphate-buffered saline). Three days later, the splenocytes obtained were fused with the parent myeloma cell line SP20. Hybrids were selected in medium containing hypoxanthine, aminopterin, and thymidine. Cloning was performed by limiting dilution, and hybridomas were screened for anti-FOCUS antibodies by an indirect binding assay with 12511 labeled sheep F(ab')2 anti-mouse immunoglobulins (specific activity, 8 to 12 ,uCi/,ug of protein; Du Pont, NEN Research Products, Boston, Mass.) as described elsewhere (41). Direct binding assays were performed with protein A affinitypurified MAb AF5 labeled with Na1251 (Amersham Corp., Arlington Heights, Ill.) by the lodo-gen (Pierce Chemical Co., Rockford, Ill.) technique to a specific activity of 12 to 16 ,uCi/,ug of protein as previously described (41). Direct binding assays were carried out in a manner similar to that of the indirect assays except that cells were incubated directly with 125I-AF5 (105 cpm in 0.2 ml of phosphate...