NRCs were prepared according to [31 (link)], and C2C12 and HEK293 cells were prepared following standard protocols. NRCs, C2C12 and HEK293 cells were transfected using Escort III (Sigma), Lipofectamine 3000 (Invitrogen) and Escort IV (Sigma), respectively, according to manufacturer’s instructions. Following incubation at 37 °C, 5% CO2 for 24–48 h for NRCs and HEK293 cells and up to 14 days for C2C12 myoblasts to allow differentiation into myotubes, cells were prepared according to [12 (link)]. Cells were stained with primary antibodies against myosin heavy chain (clone A4.1025) [9 (link)], obscurin domain O59 [61 (link)], titin Z1Z2 [15 (link)], p62/SQSTM1 (Abcam, ab41116161) and ubiquitin (Merck, FK2, 04–263) followed by Cy3 or Cy5-conjugated goat anti-rabbit or anti-mouse IgG (H + L) secondary antibodies (Jackson ImmunoResearch ML 115–165-146, 115–175-146, 111–175-144 and BioRad STAR36D549GA) and imaged on an LSM510 (Zeiss) or SP5 (Leica) confocal microscope. Antibodies were diluted 1:100 prior to incubation.
Rees M., Nikoopour R., Fukuzawa A., Kho A.L., Fernandez-Garcia M.A., Wraige E., Bodi I., Deshpande C., Özdemir Ö., Daimagüler H.S., Pfuhl M., Holt M., Brandmeier B., Grover S., Fluss J., Longman C., Farrugia M.E., Matthews E., Hanna M., Muntoni F., Sarkozy A., Phadke R., Quinlivan R., Oates E.C., Schröder R., Thiel C., Reimann J., Voermans N., Erasmus C., Kamsteeg E.J., Konersman C., Grosmann C., McKee S., Tirupathi S., Moore S.A., Wilichowski E., Hobbiebrunken E., Dekomien G., Richard I., Van den Bergh P., Domínguez-González C., Cirak S., Ferreiro A., Jungbluth H, & Gautel M. (2021). Making sense of missense variants in TTN-related congenital myopathies. Acta Neuropathologica, 141(3), 431-453.
Other organizations :
King's College London, Guy's and St Thomas' NHS Foundation Trust, King's College Hospital, Guy's Hospital, University of Cologne, University Hospital of Geneva, Queen Elizabeth University Hospital, National Hospital for Neurology and Neurosurgery, University College London, Great Ormond Street Hospital, Friedrich-Alexander-Universität Erlangen-Nürnberg, Universitätsklinikum Erlangen, University of Bonn, Radboud University Nijmegen, Radboud University Medical Center, VA San Diego Healthcare System, Gillette Children's Specialty Healthcare, University of Ulster, Belfast City Hospital, Royal Belfast Hospital for Sick Children, University of Iowa, University of Göttingen, Université d'Évry Val-d'Essonne, Inserm, Genethon (France), Université Paris-Saclay, Cliniques Universitaires Saint-Luc, Hospital Universitario 12 De Octubre, Laboratoire de Recherche Vasculaire Translationnelle, Université Paris Cité
Transfection method for NRCs (Escort III), C2C12 (Lipofectamine 3000), and HEK293 cells (Escort IV)
dependent variables
Expression of myosin heavy chain, obscurin domain O59, titin Z1Z2, p62/SQSTM1, and ubiquitin in NRCs, C2C12, and HEK293 cells
control variables
Incubation time (24-48 hours for NRCs and HEK293 cells, up to 14 days for C2C12 cells)
Cell culture conditions (37°C, 5% CO2)
Primary antibody dilution (1:100)
controls
No positive or negative controls were explicitly mentioned in the protocol.
Annotations
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