FIG. 4.

Cbl overexpression enhances the turnover of Fyn and FynY528F protein as determined by metabolic pulse-chase analysis but has no effect on the half-life of CD8-ζ. 293T cells in 100-mm tissue culture dishes were transfected with pSRαneo-CD8-ζ (0.5 μg), pAlterMAX-Fyn (0.1 pg), or FynY528F (0.1 μg) with or without the pAlterMAX-HA-Cbl expression construct (1 μg). At 48 h posttransfection, the cells were methionine starved for 1 h and then were pulse-labeled with [³⁵S]methionine for 45 min. The pulse was chased with culture medium supplemented with unlabeled methionine for the indicated times, and cell lysates were prepared. (A) Anti-Fyn immunoprecipitates (i.p.) of cell lysates were resolved by SDS-PAGE and transferred to a PVDF membrane. Labeled Fyn protein was visualized by exposing the membrane to X-ray film for 48 h (top panel). The membrane was subsequently immunoblotted with an anti-Fyn antibody (bottom panel). (B) The signals in panel A were quantified as described in Materials and Methods, and the values at various chase times are expressed as a percentage relative to the highest ³⁵S-Fyn signal. The line of best fit was calculated with the Prism program. A representative experiment is shown. This experiment was performed three times, with similar results in each case. (C) CD8-ζ protein was immunoprecipitated (i.p.) from lysates prepared by the same method as in panel A, and the labeled protein was visualized by autoradiography following transfer to PVDF (top panel, 16-h exposure). The membrane was then immunoblotted with a ζ-specific antibody (bottom panel). (D) Signals in panel C were quantified and expressed as for panel B.