Mgcl2

Following transfection with the pIRVP3IL-18HN recombinant plasmid for 72 h, 2×10⁶ H22 tumor cells were lysed in 300 µl of buffer A, consisting of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH (pH 7.4), 0.25 M sucrose, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM EDTA, 1 mM DTT, 10 mM MgCl₂ and 1 mM phenylmethylsulfonyl fluoride (all obtained from Solarbio Science & Technology Co., Ltd.) and homogenized in a Dounce homogenizer (IKA, Guangzhou, China) for 10 min. The supernatant was mixed with TNC buffer, consisting of 10 mM Tris-acetate (pH 8.0) (HD Biosciences Co., Ltd.), 0.5% Nonidet P-40 (HD Biosciences Co., Ltd.) and 5 mM CaCl₂ (Solarbio Science & Technology Co., Ltd.), and the precipitate was dissolved in buffer A to obtain the cytosol, which together with the mitochondria were stored at −80°C for subsequent western blot analysis.

The detailed procedures for α-ketoglutarate detection were carried out as previously described [53 (link)]. In short, cells were lysed with RIPA buffer (Beyotime, P0013) containing phosphatase and protease inhibitors (Beyotime, P1046), and centrifuged at 15000 × g for 5 min at 4°C. Then, the 50 μl lysates were used for detection in 450 μl assay solution (100 mM KH₂PO₄ [Solarbio, P7392], pH 7.2, 10 mM NH₄Cl [Solarbio, A7320], 5 mM MgCl₂ [Solarbio, M8161], 0.15 mM NADH, 5 U GLUD1 [Sigma-Aldrich, G2626]) and incubated for 10 min at 37°C. The absorbance at 340 nm was measured. The decreased absorbance was used to detect the level of α-ketoglutarate.