FIG. 9.

Autophosphorylation of GFP-Fes CC domain mutant proteins in TF-1 myeloid cells. TF-1 cells stably expressing each of the GFP-Fes fusion proteins indicated were washed free of GM-CSF and incubated in the presence or absence of cytokine for 48 h. Cells were collected by centrifugation and lysed, and GFP-Fes fusion proteins were precipitated with the M2 anti-FLAG monoclonal antibody. Immunoprecipitates were washed with RIPA buffer, resolved by SDS-PAGE, and immunoblotted for Fes expression with the anti-FLAG antibody (Fes) or for phosphotyrosine content with the antiphosphotyrosine monoclonal antibody PY99 (P-Tyr). This experiment was repeated twice and produced comparable results in each case; a representative blot is shown.