Extended Data Fig. 6: Generation of BCAT1 or BCAT2 Reconstituted Cell Models for Proximity Labelling.

(a) Western blot analysis of enzymes in the BCAA metabolic pathway in major tissues of WT and Bcat2^−/− mice. (b) Western blot analysis and quantification of BCKDHA and P-BCKDHA in WT and Bcat2^−/− liver samples. Results shown as mean ± SD (N = 12 WT and 11 Bcat2^−/− mice) and analysed by two-sided unpaired t-test. (c) Generation of BCAT1 or BCAT2 reconstituted HEK293T cells. Western blot analysis is shown for the mitochondrial location of mTD, BCAT1-mTD and BCAT2-mTD, and efficient biotinylation of mitochondrial proteins in the presence of doxycycline (Dox) and exogenous biotin. MT-CO2 and p70 S6 kinase (P70-S6K) were analysed as controls for the fractionated mitochondria and cytoplasm, respectively. (d) Validation of efficient biotinylation of mitochondrial proteins by proximity labelling using mTD, BCAT1-mTD or BCAT2-mTD in the presence of Dox and exogenous biotin. (e) Co-immunoprecipitation of BCAT1 and BCKDH in mouse kidney mitochondrial lysates using α-BCKDHA antibody cross-linked Dynabeads Protein A. Input or immunoprecipitated samples were blotted with α-BCKDHA and α-BCAT2 antibodies, respectively. Co-immunoprecipitation was performed in the presence or absence of additives (Val, PLP, α-KG, thiamine diphosphate and Coenzyme A), whereas co-immunoprecipitation using Dynabeads Protein A without cross-linking with α-BCKDHA antibody was performed as a negative control.

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