Fig. 7: McPGK1 promotes the binding of PGK1 and TOM40 complex.

A, B Immunoblot of TOM70/TOM40 in PGK1 immunoprecipitation (IP) eluate from mcPGK1 knockdown (A) or overexpressing (B) sphere lysate. 1% Input, 50% IgG IP eluate and PGK1 IP eluate were used for immunoblot. Typical results in left panel and protein quantitative results in right panel. C, D Immunoblot to evaluate TOM70/TOM40 levels in PGK1 IP eluate (C), or PGK1 levels in TOM70/TOM40 IP eluate (D). Primary #4 and #6 cells were used for sphere formation, and sphere lysates supplemented with gradient doses of mcPGK1 transcript were used for IP assay. E Split-APEX2 assay were established (left), followed by real-time PCR for mcPGK1 detection (middle) and Western blot for another outer mitochondrial membrane protein TOM70 (right). TOM40, PGK1 and mcPGK1 are assembled together in outer mitochondrial membrane. F mcPGK1 silenced cells were used for Split-APEX2, followed by Western blot with TOM40 and TOM70 antibodies. G Hep3B cells overexpressing WT and indicated mutant mcPGK1 were used for TRAP assay, and the enrichment of TOM70/TOM40 in TRAP eluate was evaluated via immunoblot. H Immunoblot of TOM70/TOM40 in PGK1 IP eluate. WT and mutant mcPGK1 cells were used for sphere formation, followed by IP assay with IgG control or PGK1 antibodies. n = 3 independent experiments. I, J Liver tumor cells (sample #4) overexpressing WT and mutant mcPGK1 were used for sphere formation (I) and metabolic analysis ( J). Scale bars, 500 μm. For A–D, n = 3 independent experiments; for E, n = 5 independent experiments; for I, J, n = 4 independent experiments. For D, F, G, n = 3 independent experiments with similar results. Data are shown as mean + s.d. *P < 0.05; **P < 0.01; ***P < 0.001, by two-tailed Student’s T-test. Source data are provided as a Source Data file.