Biochemical characterization of antibody ALK1. (a) Cell lysates were separated by SDS-PAGE and then subjected to Western blotting. Antibody ALK1 recognizes a protein of 80 kD in the t(2; 5)⁺ SU-DHL-1 cell line (lane 1). No protein bands are detected in the Daudi B-cell line or the Hodgkin's-derived cell lines L540, L428, HDLM-2, and KM-H2 (lanes 2 through 6). However, antibody ALK1 did detect a band of 200 kD in lysates of the human rhabdomyosarcoma Rh30 cell line (lane 7) that expresses the full-length ALK receptor protein19 and in 293T (human embryonic kidney epithelial) cells that had been transfected with a cDNA encoding the full-length ALK protein (lane 8). No bands were seen in 293T cells that had been tranfected with pcDNA3 vector only (lane 9). The smaller bands seen in the lysates from the SU-DHL-1 and 293T cells transfected with pcDNA3-ALK have been observed using polyclonal anti-ALK sera ( S.W. Morris, personal communication, March 1996) and probably represent proteolytic breakdown products of the NPM-ALK and ALK proteins, respectively. (b) Immunoprecipitates prepared from SU-DHL-1 and Daudi cell lysates using the antiphosphotyrosine antibody 4G10 were subjected to SDS-PAGE and then electroblotted. The blots were then stained with either the antiphosphotyrosine antibody 4G10 (lanes 1 and 2) or with antibody ALK1 (lanes 3 and 4). The t(2; 5)⁺ SU-DHL-1 cell line contained many phosphorylated proteins (lane 1) in contrast to only two found in the Daudi cell line (lane 2). Antibody ALK1 labeled only the prominent 80-kD phosphorylated NPM-ALK protein present in the SU-DHL-1 cells (lane 3). No bands were detected by antibody ALK1 in the Daudi cell immunoprecipitate (lane 4). The position of molecular weight standards (myosin, 205 kD; galactosidase, 116 kD; phosphorylase, 97 kD; bovine serum albumin, 68 kD; and ovalbumin, 45 kD) are indicated.