Extended Data Figure 2

a, Western blot showing protection of the ATP-2 protein from Pronase digestion upon α-KG binding in the DARTS assay. The antibody for human ATP5B (Sigma, AV48185) recognizes the epitope ₁₄₄IMNVIGEPIDERGPIKTKQFAPIHAEAPEFMEMSVEQEILVTGIKVVDLL₁₉₃ that has 90% identity to the C. elegans ATP-2. The lower molecular weight band near 20 kDa is a proteolytic fragment of the full-length protein corresponding to the domain directly bound by α-KG. b, α-KG does not affect Complex IV activity. Complex IV activity was assayed using the MitoTox OXPHOS Complex IV Activity Kit (Abcam, ab109906). Relative complex IV activity was compared to vehicle (H₂O) controls. Potassium cyanide (Sigma, 60178) was used as a positive control for the assay. Complex V activity was assayed using the MitoTox Complex V OXPHOS Activity Microplate Assay (Abcam, ab109907). c, atp-2(RNAi) worms have lower oxygen consumption compared to control (gfp in RNAi vector), P < 0.0001 (t-test, two-tailed, two-sample unequal variance) for the entire time series (2 independent experiments); similar to α-KG treated worms shown in figure 2g. d, α-KG does not affect the electron flow through the electron transport chain. OCR from isolated mouse liver mitochondria at basal (pyruvate and malate as Complex I substrate and Complex II inhibitor, respectively, in presence of FCCP) and in response to sequential injection of rotenone (Rote; Complex I inhibitor), succinate (Succ; Complex II substrate), antimycin A (AA; complex III inhibitor), ascorbate / tetramethylphenylenediamine (Asc/TMPD; cytochrome c (Complex IV) substrate). No difference in Complex I (C I), Complex II (C II), or Complex IV (C IV) respiration was observed after 30 min treatment with 800 µM of octyl α-KG, whereas Complex V was inhibited (see Fig. 2h) by the same treatment (2 independent experiments). e-f, No significant difference in coupling (e) or electron flow (f) was observed with either octanol or DMSO vehicle control. g-h, Treatment with 1-octyl α-KG or 5-octyl α-KG gave identical results in coupling (g) or electron flow (h) assays. Mean ± s.d. is plotted in all cases.