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. Author manuscript; available in PMC: 2009 Dec 4.
Published in final edited form as: Signal Transduct. 2002 Nov 7;2(1-2):29–39. doi: 10.1002/1615-4061(200205)2:1/2<29::AID-SITA29>3.0.CO;2-7

Fig. 4. The RING finger domain is required for Cbl-dependent negative regulation of Fyn-induced transcriptional activation of a SRE-luciferase reporter.

Fig. 4

A, mutations in the RING finger domain of Cbl blocks the negative regulation of SRE-luciferase activation. 293T cells were transfected with plasmids encoding the SRE-luciferase reporter (5 μg), CD8-ζ (0.5 μg) and the indicated combinations of Fyn (0.1 μg), HA-Cbl, HA-Cbl-70Z and HA-Cbl-C3AHN (1 μg) or pAlterMAX vector (−). Cells were lysed 48 hr after transfection and equal aliquots of lysate protein were used to assay the luciferase activity. The luciferase activity was expressed relative to the activity of lysates transfected with the reporter in the absence of Fyn or Cbl. Results represent the mean +/− one standard deviation of five replicate transfections. B, analysis of Fyn protein levels in transfected cells used for SRE-luciferase assay. Aliquots of lysate protein (10 μg) from two of the five replicate samples analyzed in A were resolved by SDS-PAGE and immunoblotted with anti-HA (top panel) and anti-Fyn (bottom panel) antibodies.