Fig 1: DNA-PKcs interacts with ANT2 and VDAC2 at mitochondria HeLa cells expressing Flag-tagged ANT1-3 were subjected to Co-IP using anti-Flag antibody, then analyzed by western blot as indicated.Co-IP was performed with mitochondrial fraction. Co-IP of endogenous ANT2 with anti-DNA-PKcs antibody (clone 25–4), but not control IgG.Proximity ligation assay (PLA) analysis showed that DNA-PKcs interacts with Flag-ANT2, but not Flag-ANT1 or ANT3.PLA analyses were performed by using anti-DNA-PKcs and anti-ANT2 antibodies. Results showed interactions between endogenous ANT2 and DNA-PKcs in human skin fibroblasts (HSF), HeLa cells, human HCT116 cells, and human M059K cells. No PLA signal was detected in DNA-PKcs–deficient HCT116 PKcs-/- and M059J cells.HeLa cells expressing Flag-tagged VDAC1-3 were subjected to Co-IP using anti-Flag antibody and analyzed by western blot as indicated.PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 was disrupted by knockdown of VDAC2, but not VDAC1 or VDAC3. Quantification shown on the right. ***P < 0.001. Data information: The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001).Co-IP confirmed that knockdown of VDAC2 by siRNA interrupts the interaction between Flag-ANT2 and DNA-PKcs. Source data are available online for this figure.