Fig. 6

Identification and validation of PKM1/2 as a direct binding protein of GACAT2 that influences mitochondrial function. a and b Determination of GACAT2 localization in both the cytoplasm and nucleus of PDLSCs. The cells were incubated in normal α-MEM (Nor), medium with the cementoblastic inducer EMD (EMD), or medium with both inflammatory cytokines and EMD (Infla-EMD). a Percentages of nuclear and cytoplasmic GACAT2 determined by qRT-PCR (U6 and β-actin served as the nuclear and cytoplasmic controls, respectively). b Subcellular localization of GACAT2 determined by the RNA SCOPE assay (scale bar: 100 μm). c GACAT2 expression was negatively correlated with mitochondrial inner membrane-associated gene signatures (GSEA). d The ChIRP method was applied to screen the potential proteins binding to GACAT2. e Heatmap of 15 GACAT2-binding proteins screened by ChIRP-MS assays (unique peptide ≥2, fold change >1.2); U1 probes were used as the positive control (U1), and nontargeting probes (control) were used as the negative control. f Quantification of labeled reference peptides for GACAT2-binding proteins (PRM assay, nontargeting probes were used as the negative control). g–k Product ion pattern of the selected proteins (fold change >1.5), i.e., TXN (g), AZGP1 (h), TUBA1B (i), JUP (j) and PKM1/2 (k) (PRM assay; EKLEATINELV for the TXN protein, AGEVQEPELR for the AZGP1 protein, AVFVDLEPTVIDEVR for the TUBA1B protein, NLALCPANHAPLQEAAVIPR for the JUP protein, and IYVDDGLISLQVK for the PKM1/2 protein); nontargeting probes were used as controls; different colors indicate different fragment ions from the same polypeptide, and each peptide was quantified using fragment ions. l Chromatograph of a labeled peptide (IYVDDGLISLQVK) for the PKM1/2 protein; PKM1/2 was selected for further verification because it is more closely related to mitochondrial function than AZGP1, TUBA1B and JUP (based on information arising from the UniProt Database), and TXN was excluded from further investigation due to its inappropriate chromatography (Fig. S15). m and n Confirmation of the interaction between PKM1/2 and GACAT2 by RIP assays. The PKM1/2 protein immunoprecipitated by the anti-PKM1/2 antibody was verified by immunoblotting analysis (m), and the enrichment of GACAT2 immunoprecipitated by anti-PKM1/2 antibodies was determined by RIP assays (n) (IgG antibodies were used as the control). The data are shown as the mean ± SD for n = 3; ***P < 0.001 indicate significant differences between the indicated columns