Fig. 2: TRIP12 negatively regulates FBW7 protein stability.
From: Proteasomal degradation of the tumour suppressor FBW7 requires branched ubiquitylation by TRIP12
a Western blot validation of TRIP12 as a negative regulator of endogenous FBW7 on lysates from cells transfected with two independent siRNAs targeting TRIP12 gene compared to a non-targeting control. b qRT-PCR analysis of TRIP12 and FBW7, n = 3 independent experiments. Bar graphs represent the mean ± SD of three independent experiments. c Western blots for the indicated proteins showing stabilisation of FBW7 in CRISPR/Cas9 HCT116 pools targeting TRIP12 compared to wildtype parental cells. GFP plasmid was used as transfection control and subsequently GFP and Actin blots were used as loading controls. d Schematic of CRISPR/Cas9 SAM sgRNAs targeting endogenous TRIP12 locus. e Western blot validation of TRIP12 SAM activation and negative regulation of FBW7 in HEK293T cells expressing EGFP-FBW7α. f Protein stability of epitope-tagged FBW7 in stable cell lines expressing a lentivirus-mediated GFP-targeting control (shGFP) versus shTRIP12; quantification of FBW7 protein levels normalised to actin from two independent experiments is shown in g. h Ni-NTA pulldown of ΔFbox-mutant and ubiquitylated wildtype FBW7 in stable HEK293T cells expressing the indicated shRNAs. Western blots in all panels are representative of at least three independent experiments unless otherwise stated. Source data are provided as a Source Data file.
