Ab56560

Protein gel electrophoresis and blotting analyses were performed on whole cell protein extracts obtained from patient (P) and control (C1 and C2) fibroblasts. Samples containing 30 μg protein were separated by denaturing NuPAGE 4%–12% Bis-Tris gels and transferred to nitrocellulose membrane. Immunodetection was carried out using primary antibodies against target proteins: RNase H1 (ab56560, Abcam), POLG (sc-5931, Santa Cruz), POLG2 (LS-C334882, LSBio), TWNK (gift from M Falkenberg), SSBP1 (ab74710, Abcam), TFAM (gift from RJ Wiesner), POLRMT (ab32954, Abcam), LRPPRC (ab97505, Abcam), SLIRP (ab51523, Abcam), ATAD3 (gift from JE Walker), bL12 (14795-1-AP, Proteintech), uL11 (SAB2701374, Sigma), MDDX28 (ab70821, Abcam), mS35 (16457-1-AP, Proteintech), mS18b (16139-1-AP, Proteintech), NDUFS3 (ab110246, Abcam), NDUFB8 (ab110242, Abcam), SDHA (ab14715, Abcam), SDHB (ab14714, Abcam), UQCRC1 (ab96333, Abcam), UQCRC2 (ab14745, Abcam), MT-CO1 (ab14705, Abcam), MT-CO2 (ab91317, Abcam), COX4l1 (ab14744, Abcam), ATPF1 (ab84625, Abcam), and ATPA1 (ab110273, Abcam), along with GAPDH (ab8245, Abcam), used as loading control. For quantifications, images were digitalized and analyzed with ImageJ software, and data analyses were performed in Microsoft Excel.

Western blot was used to analyze the protein extracts obtained from the fibroblast mitochondria-enriched fractions using the Mitochondria Isolation Kit for Cultured Cells (Abcam) following the manufacturer’s instructions. Mitochondrial pellets were resuspended in the resuspension reagent provided by the supplier, supplemented with a protease inhibitor cocktail (cOmplete EDTA-free, Roche). Samples were heat-denatured in loading buffer (60 mM Tris–HCl pH6.8, 20% glycerol, 20% SDS, 5% β- mercaptoethanol, 0.05% bromophenol blue) and separated by 12% SDS-PAGE. Proteins were transferred to Immun-Blot PVDF membranes (Bio-Rad) and detected using primary mouse monoclonal antibodies against human RNase H1 protein (ab56560, Abcam) and rabbit polyclonal antibodies against succinate dehydrogenase (SDHA; NB-22-14256, Neo Biotech).