Immunochemical characterization of CD148 molecule. Adult human PBMCs were surface biotinylated and CD148 molecule was immunoprecipitated by using 143-41–coupled CNBr-activated Sepharose 4B. Sample aliquots were subjected to treatment with N-endoglycosidase F (lane 3), O-endoglycosidase (lane 2), and neuraminidase (lane 1) as described in the Materials and Methods. Next, samples were analyzed on a 5% SDS-polyacrylamide gel under reducing conditions before (lane 4) and after glycanase treatment (lanes 1, 2, and 3), followed by electrophoretic transfer of proteins onto Immobilon-P. After blocking and incubating with streptavidin-peroxidase, the Western blots were developed using diaminobenzidine with cobalt enhancement.