Fig. 7: TRIP12 and UBE2S catalyse-branched ubiquitylation of FBW7.
From: Proteasomal degradation of the tumour suppressor FBW7 requires branched ubiquitylation by TRIP12
a Western blots showing levels of FLAG-FBW7 protein in HEK293T cells incubated with a control and two independent siRNAs targeting the K11-linkage-specific E2 enzyme, UBE2S. b Schematic of sequential in vitro ubiquitylation assay followed by Cezanne-mediated K11-linkage cleavage. Ubiquitylation step 1: SCFFBW7 + UBCH5 + wt-ubi; step 2: TRIP12 + UBE2S + K48R-ubi. c Western blots showing FBW7 in vitro ubiquitylation and K11-linkage addition by TRIP12. d Confirmation of K11-linkage on FBW7 by TRIP12 with a K11-specific deubiquitinase, Cezanne. e Western blots showing enrichment of K11-linked polyubiquitylated FBW7 in HEK293T cells of the indicated genotypes. f Outline of the method to detect branched ubiquitylation. g Western blots confirming branching activity of TRIP12-HECT domain in vitro, black star (*) represents a nonspecific band of unknown identity. h Schematic for FBW7 in vitro branched ubiquitylation assay. i FBW7-branched ubiquitylation by TRIP12 using the Ubi53TEV/FLAG mutant followed by TEV cleavage and blotting with FLAG-M2-HRP antibody. j Outline of the potential mechanism regulating FBW7 proteasomal degradation. See also Supplementary Fig. 5. All blots are representative of at least three independent experiments. Source data are provided as a Source Data file.
