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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. FBXL17FLAG mutants were affinity-purified from
293T cells that also expressed M YCSKP1, 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 FBXL17FLAG, as denoted on the right, M YCSKP1,
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 SCFFBXL17. Immobilized recombinant MBP, MBPF B X L 1 7,MBPFBXL17ΔCTH, or CTHMBP were incubated with (^35) S-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 FBXL17ΔCTH-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 SCFFBXL17. Unmodified and ubiquitylated KEAP1 were
detected by gel electrophoresis and autoradiography. This experiment was
performed once.