Dot-blot analysis of the recombinant GPIbα fragments, WT and M239V. Immunologic reactivity of the two recombinant proteins was evaluated using a panel of anti-GPIbα antibodies. To quantitate the amount of GPIbα antigen for dot-blot analysis, serum-free CHO-cell culture media containing either WT or M239V were first serially diluted and the amount of GPIbα-related antigen was assessed using anti-GPIbα MoAb LJ-Ibα1. LJ-Ibα1 recognizes an epitope within the first 237 residues in the N-terminal domain on GPIbα and reacts better under reducing condition. Culture media containing either WT or M239V were then appropriately diluted with nontransfected CHO-cell culture medium and the equivalent amount of antigen was applied onto the nitrocellulose membrane. After incubation with one of the first antibodies (LJ-Ibα1, GUR83/35, or anti-GPIbα^271-285 ), nitrocellulose membranes were incubated with ¹²⁵I-antimouse IgG and autoradiographed. GUR83/35 recognizes conformation-dependent epitope in the N-terminal division of GPIbα, whereas anti-GPIbα^271-285 recognizes linear epitopes created by a polypeptide sequence of GPIbα^271-285. Note that WT and M239V presented similar immunochemical reactivities to each anti-GPIbα antibody tested, regardless of the presence (R) or absence (NR) of 50 mmol/L dithiothreitol in the medium before spotting on nitrocellulose membranes. NR, not reducing; R, reducing.