Immunoprecipitation with anti-PSGL-1 mAb does not clear T-cell lysates of CLA or E-selectin ligand activity. (A) Lysates of cultured human peripheral-blood T cells expressing high levels of CLA were immunoprecipitated with anti-PSGL-1 mAb 4H10 and the products subjected to reducing SDS-PAGE. Representative Western blots stained for CLA (mAb HECA-452) are shown in panel A. All blots include high-range molecular weight markers run in the flanking lanes. Unmanipulated lysate (Original) shows a 140-kDa CLA⁺ band as previously described. IP of PSGL-1 results in recovery of 140-kDa CLA⁺ PSGL-1 (Pellet 1 is product of first IP) with complete clearance of PSGL-1 from the lysate by 3 rounds of IP (Pellet 3). The band of CLA⁺ material at 140 kDa observed in the residual soluble material after PSGL-1 IP (Supn 1 and Supn 3) indicates the presence of an additional CLA⁺ protein distinct from PSGL-1. Blots from panel A were assayed for functional E-selectin ligand activity by blot-rolling analysis (B). E-selectin ligand activity was observed on both immunoprecipitated 140-kDa PSGL-1 (Pellet 1) and the residual unknown CLA⁺ protein (Supn 3). Results shown are mean and range of 2 to 4 separate determinations on each blot and are representative of 3 similar immunoprecipitation experiments.