Extended Data Figure 9. Double knockout of BCKDK in both skeletal muscle and liver is specific in fasted and re-fed states.

a-d, Western quantification of BCKDK in fasted tissues normalized to loading control collected from n=3 WT and n=3 BCKDK double KO (Double KO) mice. e-h, Western quantification of the ratio pBCKDH/BCKDH in fasted tissues; n=3 WT and n=3 Double KO. i, Western blotting for p-ACLY and ACLY in fasted livers. 14-3-3 is the loading control; n=6 WT and n=6 Double KO. j, Western quantification of the ratio p-ACLY/ACLY from the blot in i. k, Body weights of n=10 WT and n=10 Double KO mice on HFD. l, Lean mass of n=8 WT and n=8 Double KO mice on HFD. m, Ratio of 3-HIB/valine, and n, Concentration of 3-HIB in plasma in mice fasted for 5-hr or re-fed for 2-hr after an overnight fast; n=10 WT and n=10 Double KO mice fed HFD. o-r, Western quantification of BCKDK in re-fed tissues normalized to loading control; n=3 WT and n=3 Double KO. s-v, Western quantification of the ratio pBCKDH/BCKDH in re-fed tissues; n=3 WT and n=3 Double KO. w, Western blotting for p-ACLY and ACLY in re-fed livers. 14-3-3 is the loading control; n=6 WT and n=6 Double KO. x, Western quantification of the ratio p-ACLY/ACLY from the blot in w. y, Body weights of n=8 BCKDK double KO + vehicle (Veh)-treated and n=8 BCKDK double KO + BT2-treated mice fed HFD. Mice used for these experiments were aged 13–23 weeks and fed HFD for 6–7 weeks. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: *p<0.05 and ***p<0.001. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures.