Figure 4.

In vivo and in vitro ubiquitination of TSC2 by FBW5–DDB1–CUL4 ligase. (A) CUL4, DDB1, and FBW5 are required for TSC2 ubiquitination in vivo. U2OS cells were cotransfected with plasmids expressing HA-TSC2 and Flag-ubiquitin and siRNA targeting CUL4A/B, DDB1, or FBW5. Cells were lysed 24 h after transfection, and TSC2 ubiquitination was analyzed by immunoprecipitating HA-TSC2 and blotted with an antibody recognizing Flag. (B) In vitro ubiquitination of TSC2 by FBW5 and CUL4A. 293T cells were cotransfected with plasmids expressing the indicated myc-tagged proteins. Individual protein complex was eluted by incubating with molar excess myc antigen peptide and mixed as indicated with substrate GST-TSC2 in the presence of E1, E2, ubiquitin, and ATP. Reaction mixtures were incubated for 30 min, terminated by boiling directly in SDS sample buffer, resolved by SDS-PAGE, and immunoblotted with indicated antibodies. (C) Drop-out assay was carried out as described in B to determine the specificity of in vitro TSC2 ubiquitination.