Additional file 2 of A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics
Creators: name: Kienzle, Laura
name: Bettinazzi, Stefano
name: Choquette, Thierry
name: Brunet, Marie
name: Khorami, Hajar Hosseini
name: Jacques, Jean-François
name: Moreau, Mathilde
name: Roucou, Xavier
name: Landry, Christian R.
name: Angers, Annie
name: Breton, Sophie
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 2: Figure S1. Using the PepQuery tool to interrogate spectrums from published proteome datasets. Figure S2. Putative post-translational modifications of MTALTND4. O-Glyc: putative O-glycosylation; P: putative phosphorylation. PTM prediction were done using webservers reviewed by [75], only scores with 80% probabilities were retained. Figure S3. Specificity of the anti-MTALTND4 antibody and detection of putative post-translational modificationsof MTALTND4. A. Complete western blot of the antibody on a cell lysate. B. Specificity of anti-MTALTND4 and PTMs. Deglyc: deglycosylationnon-treated cells,treated cells and fetuin positive control are shown); Dephosphos: dephosphorylationnon-treated cells andtreated cells are shown). C. Apparent molecular weight of MTALTND4 exposed to different concentrations of denaturing agent β-Mercaptoéthanol and different heating times at 95˚C. A = 5 minutes, B = 30 minutes and C = 3 hours. 1 = 0% β-Mercaptoethanol, 2 = 5% β-Mercaptoethanol and 3 = 10% β-Mercaptoethanol. D. Self-association of MTALTND4: 3 µg of synthetic peptide were incubated at 30 °C for 10 min in the presence of 0%, 0.5% and 1% paraformaldehyde, quenched with LSB, separated by tricine-SDS-PAGE and visualized with Coomassie Imperial Protein stain. Arrowheads indicate main cross-linked products. Figure S4. Comparison of MTALTND4 peptide encoded in mitochondrial DNAand hypothetically in nuclear DNA that have been transferred from mtDNAthrough evolution. Only sequences with &gt;50% identities and complete without stop codons are shown. Figure S5. Multiple MTALTND4 peptide sequence alignments. A. In 15 mammal species: human, chimpanzee, bonobo, gorilla, orangutan, mouse, rat, naked mole rat, dog, cow, zebrafish, lion, bear, horse, dolphin. B. In the genus Homo: Homo heidelbergensis; Homo sapiens neanderthalensis; Homo sapiens neanderthalensis; Homo sapiens neanderthalensis; Homo sapiens neanderthalensis; Homo sapiens neanderthalensis; modern human, chimpanzee. *Indicates a stop codon. Figure S6. Endogenous MTALTND4 detected in HeLa cells by mass spectrometry. Unique mass spectrometry MTALTND4-derived peptides detected in HeLa cells lysates. Probabilities = 100%. Figure S7. Effect of MTALTND4 on cell viability. HeLa and HEK-293T cells were cultured in low glucose DMEM with 0.1 µM, 10 µM or 30 µM MTALTND4 peptide or waterfor 24h or 48h and assessed for cell viability using the alamarBlue assay. Figure S8. GST pull-down assay indicating that MTALTND4 and Complement component 1 Q interact. Western blot probed with anti-C1qbp antibodies. Lane 1: pull-down products from GST bound glutathione beads. Lane 2: pull-down products from GST-MTALTND4–bound glutathione beads. Lane 3: HeLa cells lysate. Lane 4: pull-down products from GST bound glutathione beads. Lane 2: pull-down products from GST-MTALTND4–bound glutathione beads. Lane 3: HEK-293T cells lysate. Figure S9. Original uncropped blots for MTALTND4.