Regulation of PGK1 succinylation and its effect on glycolysis. (A) The lysine-succinylation (pan-Ksu) levels after treatment with different concentrations of succinate were detected by WB. (B) The succinyl-CoA levels after treatment with different concentrations of succinate were detected by ELISA. (C) MS identification of K191, K192 succinylation of PGK1 in TBD0220 cells transduced with shP4HA1 lentiviral vector. (D) Analysis of the conserved amino acid sequence of PGK1 using the Consurf coloring scheme. Maroon and cyan indicate high and low conservations, respectively. (E) The co-IP analysis of lysine-succinylation level by immunoprecipitating with anti-PGK1 antibody under P4HA1-OE or KD condition. (F) Glioma cells were transfected with WT, 2KR, or 2KQ of Flag-PGK1. The lysine-succinylation levels of immunoprecipitated Flag-PGK1 (WT and mutants) were assessed by WB. (G and H) The ECAR was measured in U87MG (G) and TBD0220 (H) cell lines after transducing with vector control, Flag-PGK1 WT, 2KR or 2KQ plasmid. (I) The ECAR was measured in glioma cell lines with Flag-PGK1-WT lentivirus after treating with succinate supplement for 24 h. (J) WB analysis of PGK1 levels in glioma cells treated with CQ (25 μM), MG132 (10 μM), respectively. (K) WB analysis of PGK1 levels in glioma cells treated with MG132 (10 μM) with or without succinate supplement. (L) WB analysis of the Flag-PGK1 protein degradation after treatment with CHX at the indicated time. (M) Co-IP analysis with anti-Flag antibody in U87MG and TBD0220 cell lines transduced with vector control, Flag-PGK1 WT, 2KR, or 2KQ vector, followed by immunoblotting with anti-ubiquitin antibody. Succ indicated succinate. 2KR, both Lys191 and Lys192 residues were replaced by Arg, and 2KQ, both Lys191 and Lys192 residues were replaced by Gln. CQ, 25 μM. MG132, 10 μM. The concentration of succinate added to U87MG and TBD0220 cells was 10 mM and 0.8 mM, respectively. Error bars represent mean ± SEM; n = 3–5 independent experiments. P-values are based on one-way ANOVA. ^*P < .05, ^**P < .01, ^***P < .001, ^****P < .0001 and n.s. indicated no significant difference.