Comparison of GP Ib, GP Ibα, and GP Ibβ from transfected CHO cells and human platelets. (A) GP Ib and GP Ibα from CHO cells and platelets of 2 donors (plt1, plt2) that had been resolved in a 5% Tris-glycine SDS gel under either nonreducing (N.R.) or reducing (R.) conditions and blotted by WM23. (B) GP Ibβ from CHO cells and platelets that had been resolved in a 4% to 12% Bis-Tris SDS gel under reducing (R.) conditions and blotted by Gi27. (C) Analysis of genomic Ibα gene sequences from the 2 donors. Genomic DNA was prepared from the buffy coat, and the VNTR-containing gene fragment was amplified by PCR along with various Ibα expression vectors. The amplified fragments were resolved in a 2.3% agarose gel. The 100 bp (base pair) DNA ladder was shown on the left. Four pcDNA-based vectors expressing GP Ibα with type A-D VNTR were included as standards on the right. (D) GP Ibα (type C VNTR) from platelets (plt2) and CHO cells before and after deglycosylation. Both Ibα were coimmunoprecipitated with SZ2 antibody, eluted from protein G-agarose beads, treated with a mixture of glycanases, resolved in a 5% Tris-glycine SDS gel under reducing conditions, and blotted by WM23.