Localization of the CrkL:CD34i_fullinteraction domain on CD34.

Competitive inhibition of CrkL:CD34i_full association by intracellular CD34 peptide. To localize the putative CD34 domain that serves to interact with CrkL, competitive inhibition studies were performed using synthetic peptides corresponding to highly conserved domains within the intracellular tail of CD34. KG1a cell lysates (4 × 10⁶ cells/point) were precleared with GST-Sepharose. Prior to pull-down with GST-CD34i_full fusion protein, certain samples were incubated 30 minutes with 200 μg/mL CD34 peptide No. 1 or No. 2, as indicated. Following SDS-PAGE and transfer to nitrocellulose membrane, proteins present in GST-CD34i_full–precipitated material were immunoblotted (WB) with rabbit anti-CrkL antibody. Binding of CrkL was competed with peptide No. 2 (RRSWSPTGER), thereby demonstrating that CrkL specifically interacts with CD34 (directly or indirectly) at a CD34 domain corresponding to this sequence. The location of this intracellular sequence corresponds to a region near the transmembrane domain that is encoded in both full-length and truncated CD34 proteins and that is entirely conserved between human, murine, and canine species. Peptide No. 1 was not able to inhibit CrkL, suggesting that interactions with CrkL do not occur at the corresponding CD34 domain that is nearer to the carboxyl tail of CD34.