Western Blot and Immunolocalization of RAP2.3

Western blots were carried out as previously described^{26 (link)}. Primary antibodies used were anti-HA (Sigma, H3663-200UL; 1:1000 dilution), anti-FLAG (Sigma, F1804-200UG, 1:2000 dilution) and secondary antibody Goat anti-Mouse IgG1, HRP from Thermo Fisher Scientific, PA1 74421, (1:10000 dilution). Full scan uncropped Western blots are provided (Source Data 1). Immunolocalization of RAP2.3 was carried as described^{70 (link)}. Four-day-old seedlings containing the 35S:RAP2.3^3XHA transgene were treated for 3 h with 50 μM bortezomib or DMSO (control). Anti-HA primary antibody (Roche, Lewes, UK) and Alexa-Fluor-488-coupled anti rat secondary antibody (1:200 dilution) (Molecular Probes, Carlsbad, CA) were used for detection. Seedlings were counter stained using Propidium Iodide and visualised using confocal microscopy.

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Zubrycka A., Dambire C., Dalle Carbonare L., Sharma G., Boeckx T., Swarup K., Sturrock C.J., Atkinson B.S., Swarup R., Corbineau F., Oldham N.J, & Holdsworth M.J. (2023). ERFVII action and modulation through oxygen-sensing in Arabidopsis thaliana. Nature Communications, 14, 4665.

Publication 2023

H3663-200UL F1804-200UG Goat anti-Mouse IgG1, HRP Anti-HA primary antibody

Alexa fluor 488 Anti antibody Antibodies Antibody igg1 Bortezomib Confocal microscopy Dilution Dmso Goat Molecular probes Mouse Propidium iodide Scan Seedlings Transgene Western blots

Corresponding Organization : University of Nottingham

Other organizations : University of Oxford, Institut de Biologie Paris-Seine, Sorbonne Université, Centre National de la Recherche Scientifique

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Variable analysis

independent variables

Treatment with 50 μM bortezomib or DMSO (control)

dependent variables

Immunolocalization of RAP2.3

control variables

Four-day-old seedlings containing the 35S:RAP2.3 3XHA transgene

controls

Positive control: DMSO treatment
Negative control: Not specified

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