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. 2023 Apr 8;30(6):1517–1532. doi: 10.1038/s41418-023-01157-6

Fig. 4. GLTC-induced succinylation of LDHA at K155 increases its enzymatic activity in PTC cells.

Fig. 4

A, B K1 cells were cotransfected with HA-LDHA and pCDNA-GLTC (or the empty vector control) for 48 h (A), (n = 3). TPC-1 cells were cotransfected with HA-LDHA and si-GLTC#1 (or si-NC as the negative control siRNA) for 48 h (B), (n = 3). Cell lysates were immunoprecipitated with HA-LDHA affinity gel. LDHA succinylation and protein levels were analysed by immunoblotting using the indicated antibodies. C K1 cells were transfected with HA-LDHA and pCDNA-GLTC (or the empty vector control) for 48 h, (n = 3). Cell lysates were immunoprecipitated with HA-LDHA affinity gel. LDHA acetylation (Ac), malonylation (Mal), and glutarylation (Glu) were analysed by immunoblotting using the indicated antibodies. D Identification of succinylated LDHA peptides by mass spectrometry in K1 cells transfected with the empty vector control (vector) or pCDNA-GLTC plasmid (GLTC). E LC‒MS/MS spectrum of the K155-succinylated peptide of LDHA. F Alignment of protein sequences surrounding K155 of LDHA from different organisms. Xl: Xenopus laevis, frog; Mm: Mus musculus, mouse; Ss: Sus scrofa, pig; Rn: Rattus norvegicus, Norway rat; Hs: Homo sapiens, human. G HA-LDHAWT, HA-LDHAK155R or HA-LDHAK155E proteins were expressed in TPC-1 cells and purified by immunoprecipitation, (n = 3). LDHA enzymatic activity was measured and normalised to the protein level. H, I Western blot analysis of K155-succinylated LDHA and endogenous LDHA levels in TPC-1 cells transfected with si-GLTC#1/2 (or si-NC as the negative control siRNA) and in K1 cells transfected with pCDNA-GLTC (or the empty vector control), (n = 3). J, K TPC-1 cells were cotransfected with si-GLTC#1 (or si-NC as the negative control siRNA) and HA-LDHAWT, HA-LDHAK155R or HA-LDHAK155E for 48 h (J), (n = 3). K1 cells were cotransfected with pCDNA-GLTC (or the empty vector control) and HA-LDHAWT, HA-LDHAK155R or HA-LDHAK155E for 48 h (K), (n = 3). LDHA was immunoprecipitated. Succinylation of K155 was evaluated by western blotting, and LDHA activity was assayed. The P value in (G) was determined by one-way ANOVA. The P values in (J and K) were determined by two-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01. NS not significant.