Detection of potential CrkL:CD34 interactions.
Endogenous CrkL and CD34 proteins associate in vivo. Coimmunoprecipitation (IP) studies were performed to confirm CrkL:CD34 interaction. KG1a cell lysates (8 × 106cells/point) were precleared with protein A–Sepharose, and proteins tightly associated with CD34 protein were coimmunoprecipitated with CD34 antibody (HPCA-1; 5 μg). Precipitated proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. Filters were cut according to protein size and immunoblotted (WB) either with rabbit anti-CrkL antibody or with mouse anti-CD34 antibody. CrkL and CD34 proteins were detected in the same sample coimmunoprecipitated with anti-CD34 but not in the sample precipitated with isotype-matched control immunoglobulin (MOPC 21). These results demonstrate that endogenous CrkL and CD34 proteins are associated, either directly or indirectly within a protein complex.
