Article
Extended Data Fig. 9 | The N-terminal β-strand is important for BTB
complex formation and recognition. a, Chimeric KLHL12 with β-strand and
adjacent dimer interface residues of KEAP1, but not wild-type KLHL12, forms
heterodimers with KEAP1 in vivo. 293T cells were transfected with KLHL12FLAG
(wild-type or chimaera) and KLHL12HA or HAKEAP1, as indicated. KLHL12FLAG
variants were immunoprecipitated and bound proteins detected by gel
electrophoresis and western blotting. This experiment was performed once.
b, BTB heterodimers are inactive in signalling. 293T cells were transfected with
FLAG-tagged wild-type KLHL12, chimeric KLHL12, or wild-type KEAP1. The
FLAG-tagged BTB proteins were affinity-purified and bound endogenous
targets of KLHL12 (SEC31, PEF1, ALG2) or KEAP1 (NRF2) were detected by
western blotting. This experiment was performed once. c, A chimeric KLHL12
that contains helix and β-strand residues of KEAP1 efficiently heterodimerizes
with KEAP1, yet fails to bind substrates of either KLHL12 or KEAP1. KLHL12FLAG,
chimeric KLHL12FLAG or KEAP1FLAG were affinity-purified from 293T cells and
bound proteins were determined by CompPASS mass spectrometry. This
experiment was performed in two technical replicates with similar results.