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. 2000 Feb;20(3):851–867. doi: 10.1128/mcb.20.3.851-867.2000

FIG. 2.

FIG. 2

Overexpression of Cbl decreases the phosphorylation of in vivo Fyn substrates, reduces the steady-state pools of Fyn proteins, and inhibits the Fyn- or FynY528F-dependent transactivation of an SRE-luciferase reporter. (A) 293T cells were transfected with the indicated amounts of pAlterMAX-Fyn or FynY528F and 0.5 μg of pSRαneo-CD8-ζ, with or without the pAlterMAX-HA-Cbl expression construct (1 μg). Cell lysates were prepared 48 h posttransfection, and 15-μg aliquots of cell lysate were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with the indicated antibodies. Fyn protein levels were determined as described in Materials and Methods. This experiment was performed three times, with similar results in each case. (B) 293T cells were transfected with the SRE-luciferase reporter construct (5 μg), together with pSRαneo-CD8-ζ (0.5 μg), pAlterMAX-Fyn or FynY528F (0.1 μg), and pAlterMAX-HA-Cbl (1 μg) expression plasmids as indicated. At 48 h posttransfection, the cells were lysed and equal amounts of protein were used to assay luciferase activity. Results are shown as the mean and 1 standard deviation (SD) based on five replicate transfections, and are expressed relative to the luciferase activity of the mock transfectant, which was arbitrarily set at 1. (C) Fyn and Cbl protein expression from panel B was determined by separating 7.5-μg aliquots of three replicates by SDS-PAGE, transferring them to a PVDF membrane, and immunoblotting with the appropriate antibodies. Relative Fyn protein levels are shown above the Fyn immunoblot. (D) 293T cells were transfected with pAlterMAX-FynY528F (0.1 μg), pSRαneo-CD8-ζ (0.5 μg), and the SRE-luciferase (5 μg) constructs, along with the indicated amounts of pAlterMAX-HA-Cbl. At 48 h posttransfection, the cells were lysed and SRE-luciferase activity was measured as in panel B. Results are shown as the mean and 1 SD based on five replicate transfections and are expressed relative to the luciferase activity of the mock transfectant, which was arbitrarily set at 1. (E) Aliquots (15 μg) of the cell lysates used in panel D were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with either anti-HA (top panel) or anti-Fyn (bottom panel) antibodies. The relative Fyn protein levels are shown above the Fyn immunoblot. Tyr(P), phosphotyrosine.