EPO−/−, WT controls, and HEK-293 cells transfected with hEPO overexpression vector49 (link) were subjected to immunoblot analysis, as described previously,62 (link) using a monoclonal mouse anti-EPO antibody (1:1,000; MAB2871, R&D systems, Abingdon, UK)63 (link) and a mouse anti-GAPDH antibody (1:1,000; sc-47724, Santa Cruz Biotechnology, Texas, USA). Goat anti-mouse IgG (H + L) Cross-Absorbed Alexa Fluor 680 (1:5,000; A21057, Invitrogen, Massachusetts, USA), was used as the secondary antibody. Membranes were visualized on the LI-COR Odyssey CLx system (LI-COR Biotechnologies, Nebraska, USA). Images were converted to grayscale using Image Studio Lite version 5.2.5 (LI-COR Biotechnologies, Nebraska, USA).
Harlow C.E., Gandawijaya J., Bamford R.A., Martin E.R., Wood A.R., van der Most P.J., Tanaka T., Leonard H.L., Etheridge A.S., Innocenti F., Beaumont R.N., Tyrrell J., Nalls M.A., Simonsick E.M., Garimella P.S., Shiroma E.J., Verweij N., van der Meer P., Gansevoort R.T., Snieder H., Gallins P.J., Jima D.D., Wright F., Zhou Y.H., Ferrucci L., Bandinelli S., Hernandez D.G., van der Harst P., Patel V.V., Waterworth D.M., Chu A.Y., Oguro-Ando A, & Frayling T.M. (2022). Identification and single-base gene-editing functional validation of a cis-EPO variant as a genetic predictor for EPO-increasing therapies. American Journal of Human Genetics, 109(9), 1638-1652.
Anti antibody Anti igg Antibody GapdhGoat Hek 293 cells Immunoblot analysis Membranes Mouse
Corresponding Organization : University of Exeter
Other organizations :
University Medical Center Groningen, University of Groningen, National Institute on Aging, Data Tecnica International (United States), National Institutes of Health, University of North Carolina at Chapel Hill, AbbVie (United States), University of California, San Diego, North Carolina State University, Azienda Sanitaria di Firenze, University Medical Center Utrecht, GlaxoSmithKline (United States)
HEK-293 cells transfected with hEPO overexpression vector
dependent variables
Immunoblot analysis of EPO and GAPDH protein levels
control variables
Experimental procedures described previously in reference 62
controls
Positive control: WT controls
Negative control: EPO−/−
Annotations
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