Extended Data Fig. 5: Validation of SKP1/FBXL17-BTB structure through FBXL17 mutations.

a, Single mutations of FBXL17 rarely affect co-translational recognition of KEAP1 in cells. FBXL17^FLAG mutants were affinity-purified from 293T cells that also expressed ^MYCSKP1, ^HAKEAP1, and dominant negative CUL1 to prevent degradation of the BTB protein. Bound proteins were detected by gel electrophoresis and western blotting. Red, mutations that abolish binding to FBXL17; orange, mutations that weaken binding to FBXL17; green, wild-type FBXL17; black: mutations that had no effect on KEAP1 binding. This experiment was performed once. b, Single mutations of FBXL17 rarely interfere with the proteasomal degradation of KEAP1. 293T cells were transfected with ^HAKEAP1 and either wild-type or mutant FBXL17^FLAG, as denoted on the right, ^MYCSKP1, and dominant-negative CUL1 (dnCUL1), as indicated. The abundance of KEAP1 was monitored by gel electrophoresis and αHA-Western blotting. This experiment was performed once. c, The CTH is required, but not sufficient, for BTB recognition by SCF^FBXL17. Immobilized recombinant MBP, ^MBPFBXL17, ^MBPFBXL17^ΔCTH, or CTH^MBP were incubated with ³⁵S-labelled fused dimers of the BTB domains of KLHL12 (green) and KEAP1 (orange). Bound proteins were detected by gel electrophoresis and autoradiography. This experiment was performed once. d, The CTH is required for in vitro ubiquitylation of mutant KEAP1. Recombinant FBXL17-SKP1 or FBXL17^ΔCTH-SKP1 were added to reticulocyte lysate after the synthesis of either wild-type or mutant KEAP1. Reticulocyte lysate contains all other components required for in vitro ubiquitylation through SCF^FBXL17. Unmodified and ubiquitylated KEAP1 were detected by gel electrophoresis and autoradiography. This experiment was performed once.