Extended Data Figure 5: Biochemical characterization of the substrate adaptor role of KBTBD8.
From: Cell-fate determination by ubiquitin-dependent regulation of translation
a, Domain structure of KBTBD8, including the residues mutated to generate ubiquitylation- (Y74A) and substrate-binding-deficient KBTBD8 (F550A, W579A). b, Effects of point mutations in predicted KELCH domain loops on binding of KBTBD8 to candidate substrates were determined by affinity purification and western blot analysis. c, Effects of point mutations in BTB domain on binding of KBTBD8 to CUL3 were determined by affinity purification and western blotting. Dimerization of Flag–KBTBD8 with KBTBD8–HA was analysed in the same experiment to provide a folding control. d, Binding of recombinant CUL3 to immobilized recombinant MBP–KBTBD8 variants was analysed by Coomassie. e, Binding of in vitro-transcribed/translated 35S-NOLC1 to immobilized recombinant KBTBD8 variants was analysed by autoradiography. f, Binding of in vitro-transcribed/translated 35S-TCOF1 to immobilized recombinant KBTBD8 variants was analysed by autoradiography. g, Endogenous β-arrestin proteins in reticulocyte lysates binds immobilized, recombinant KBTBD8, as detected by western blot analysis. h, 293T cells were transfected with control- or β-arrestin 1/2-siRNAs and reconstituted with Flag–KBTBD8. Binding of KBTBD8 to endogenous TCOF1 and NOLC1 was analysed by anti-Flag affinity purification and western blot analysis. i, Ubiquitylation of HA–TCOF1 in 293T cells depleted of β-arrestin 1/2 and reconstituted with KBTBD8 was determined after denaturing Ni-NTA purification by western blotting as described above. j, Ubiquitylation of HA–NOLC1 was detected in 293T cells depleted of β-arrestins and reconstituted with KBTBD8, as described above.
