RBOHD | Respiratory burst oxidase homolog protein D
AS15 2962 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Solanum lycopersicum
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Total proteins were isolated from 10 or 12 seedlings of 7-day-old Arabidopsis thaliana seedlings of Col-0 (wild-type) and rbohd null mutant (Nühse et al., 2007). 16 µl of total proteins were denatured at 65°C for 5 min, separated on an 8 % SDS-PAGE. and transferred for 70 min at 55V using a tank transfer system to nitrocellulose membrane. Blots were blocked with 1xPBS+0.1% Tween 20 + 5 % milk (PBS-T) or TBS + 0.1 % Tween 20 (TBS-T) + 5 % milk for 2 h at room temperature (RT) with agitation. Primary antibodies were diluted as indicated to 1: 2,500 and 1: 5,000 respectively and incubated ON at 4ºC with agitation in 1xPBS-T pr TBS-T + 5 % milk. The primary antibody solutions were decanted, and the blots were washed 3 times (7 min. each) in 1xPBS-T or TBS-T at RT with agitation. Blot was incubated with secondary antibody goat anti-rabbit HRP conjugated (AS09 602, Agrisera) diluted to 1: 20, 000 and developed with chemiluminescent detection reagent, accodring to manufacture's recommendations. Exposure time was 2 min. for X-ray film. As a control part of the same membrane was incubated with anti-CNX1/2 antibodies (AS12 2365, Agrisera).
Nühse et al. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J. 2007 Sep; 51(5): 931–940.
Courtesy of Nga Nguyen, Kelly Mason and Antje Heese (University of Missouri- Columbia, MO, USA
Lee et al. (2020). Regulation of reactive oxygen species during plant immunity through phosphorylation and ubiquitination of RBOHD. Nat Commun. 2020 Apr 15;11(1):1838. doi: 10.1038/s41467-020-15601-5.
Jedelská et al. (2019). Tomato Root Growth Inhibition by Salinity and Cadmium Is Mediated By S-Nitrosative Modifications of ROS Metabolic Enzymes Controlled by S-Nitrosoglutathione Reductase. Biomolecules. 2019 Aug 21;9(9). pii: E393. doi: 10.3390/biom9090393.
Otulak-Kozie? et al. (2019). The Respiratory Burst Oxidase Homolog D (RbohD) Cell and Tissue Distribution in Potato–Potato Virus Y (PVYNTN) Hypersensitive and Susceptible Reactions. Int. J. Mol. Sci. 2019, 20(11), 2741. (innunofluorescence)
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