Detection of GST-CD34i_full–associated proteins.

To screen for potential CD34-associated proteins, logarithmically growing KG1a cells were adjusted to 6 × 10⁵/mL and loaded with ³⁵S-methionine/cysteine for 16 hours to label intracellular proteins. Cells were stimulated with 15 μg/mL CD34 antibody (“CD34”; 9C5), Ig control (“Ig”; MOPC 21), 200 nM TPA, or 0.1% DMSO control. After 30 minutes at 37°C, cells were washed, lysed, and ³⁵S-labeled proteins were pulled down with 20 μg GST-CD34i_full fusion protein (“CD34i”) or control GST protein (“GST”). Shown is an autoradiograph indicating inducible precipitation of an unidentified 45-kd band with GST-CD34i_full fusion protein after stimulation of KG1a cells with anti-CD34 or TPA. Also shown is a 36-kd protein band constitutively associated with GST-CD34i_full. A faint 39-kd protein band constitutively associated with GST-CD34_ifullis visible below the 45-kd band. Results demonstrate that under these assay conditions, both inducible and constitutive protein interactions with GST-CD34 can be detected. To specifically identify any known signaling intermediates present in material precipitated with GST-CD34i_full, in a parallel experiment nitrocellulose filters were immunoblotted with anti-CrkL antibody (results shown here) or with antibodies corresponding to certain other adapter proteins (Figure 7). The data indicate that CrkL protein was precipitated with the GST-CD34i_full fusion protein, and not with GST control, and indicate interaction (direct or indirect) between CD34 and CrkL proteins in vitro. WB indicates Western immunoblot analysis.