Western blotting was performed as described previously (Oppermann et al. 2019 (link)). Briefly, 30 µg of protein was separated on a 4–20% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) gradient gel (Bio-Rad, Munich, Germany) and was transferred to a PVDF (polyvinylidene difluoride) membranes (Low Fluorescence Membrane Opti Blot, Abcam, Cambridge, UK). The primary antibodies used were: mouse anti-DOCK4 (Santa Cruz Biotechnology, Heiderlberg, Germany #sc-100718 1:500), rabbit anti-GAPDH antibody (Cell Signaling; #2118 1:5000) and mouse anti-ACTB (Novus Biologicals #NB600-501, 1:5000). The secondary antibodies employed (red fluorescent IRDye 680RD goat anti-mouse and green fluorescent IRDye 800CW goat anti-rabbit; both diluted 1:10,000 in TBST) were purchased from LI-COR (LI-COR Biosciences, Lincoln, USA). Membranes were scanned using an Odyssey Imaging System (LI-COR, Bad Homburg, Germany), and band intensities were determined by the Image Studio 5 software (LI-COR).
Herbst C., Bothe V., Wegler M., Axer-Schaefer S., Audebert-Bellanger S., Gecz J., Cogne B., Feldman H.B., Horn A.H., Hurst A.C., Kelly M.A., Kruer M.C., Kurolap A., Laquerriere A., Li M., Mark P.R., Morawski M., Nizon M., Pastinen T., Polster T., Saugier-Veber P., SeSong J., Sticht H., Stieler J.T., Thifffault I., van Eyk C.L., Marcorelles P., Vezain-Mouchard M., Abou Jamra R, & Oppermann H. (2024). Heterozygous loss-of-function variants in DOCK4 cause neurodevelopmental delay and microcephaly. Human Genetics, 143(3), 455-469.
Other organizations :
Leipzig University, Evangelisches Krankenhaus Bielefeld, Bielefeld University, Centre Hospitalier Régional Universitaire de Brest, University of Adelaide, Génétique Médicale & Génomique Fonctionelle, Tel Aviv Sourasky Medical Center, Friedrich-Alexander-Universität Erlangen-Nürnberg, University of Alabama at Birmingham, HudsonAlpha Institute for Biotechnology, Barrow Neurological Institute, University of Arizona, Phoenix Children's Hospital, Université de Rouen Normandie, Inserm, Invitae (United States), Helen DeVos Children's Hospital, Institut du Thorax, Nantes Université, Centre National de la Recherche Scientifique, Children's Mercy Hospital, Seoul National University
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