Ab97505

The mouse monoclonal antibody against ECD was previously described (31 (link), 39 (link)). The antibodies used in these studies include those against PRPF8 (ab79237), DDX39A (ab96621), LRPPRC (ab97505), and ALY (ab202894) and were from Abcam. Antibodies against CRM1 (46249) and ErbB2 (2242S) were purchased from Cell Signaling. For immunofluorescence staining, we used anti-ECD (catalog number  HPA006465; Sigma-Aldrich) and anti-DDX39 (catalog no. sc-271395; Santa Cruz Biotechnology) antibodies; secondary antibodies tagged with Alexa Fluor 488 (A32731 and A32723) and Alexa Fluor 568 (A-11011 and A-11031) and anti-FLAG antibody (F3165) were from Sigma-Aldrich.

The tissue microassay was stained for immunohistochemical analysis. Microarray was baked at 60°C for 2 h, deparaffinization with xylene, and then rehydrated after being washed three times in 1× phosphate-buffered saline (PBS). Then, rehydrated microarray was incubated with 3% hydrogen peroxide for 10 min in methanol to inactivate endogenous peroxidase activity and then blocked using 2.5% bovine serum albumin dissolved in PBS against nonspecific binding sites for 30 min at room temperature. Then anti-LRPPRC antibody (diluted in 1:200; Cat. No: ab97505; Abcam, Cambridge, England) was added for overnight incubation at 4°C. Incubated microarray was then rinsed three times in ice-cold PBS and incubated with a horseradish-peroxidase-conjugated antibody (diluted in 1:5,000; Cat. No: ab7090; Abcam) for 1 h at room temperature. The microarray was developed then with 3, 3′-diaminobenzidine solution for 2–5 min, washed briefly in running water, and imaged under a microscope (Olympus BX51; Olympus, Japan).

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.