Deletion of the GPIbα 580-590 sequence does not interfere with Ser609 phosphorylation. (A) Dot blots of BSA-CYSGHSL or BSA-CYSGH(pS)L (peptides corresponding to the nonphosphorylated and phosphorylated C-terminus of GPIbα) with IgG against either the phosphorylation-independent (anti-Ibα+/-P) or phosphorylated (pSer609) C-terminal peptide of GPIbα. Blots were visualized using standard ECL substrates. (B) Western blots (WB) of whole platelet lysates analyzed by SDS-PAGE under reducing (R) or nonreducing (NR) conditions and probed with anti-pS609 (pSer609). The major band at 135 kDa corresponds to GPIbα (disulfide-linked to GPIbβ, nonreduced); the band at approximately 20 kDa reduced or approximately 50 kDa nonreduced represents the membrane-associated calpain digestion fragment of GPIbα (minus the soluble extracellular glycocalicin fragment), which is linked to GPIbβ when nonreduced. (C) Triton X-100 lysates of GPIb-IX CHO cells were separated by 4% to 15% SDS-PAGE and immunoblotted with the pSer609 polyclonal antibody. Phosphorylated GPIbα was detected in cells expressing the wild-type complex or a complex containing the Δ580-590 deletion but not in cells lacking the entire GPIbα intracellular domain.