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. 2023 Oct 7;14:6262. doi: 10.1038/s41467-023-41824-3

Fig. 4. GmGA2ox-like is ubiquitinated and degraded by the proteasome.

Fig. 4

a In vivo ubiquitination of GmGA2ox-like as detected by co-immunoprecipitation (Co-IP) assay. b Identification of the ubiquitination sites essential for GmGA2ox-like ubiquitination. c Ubiquitination of GmGA2ox-like by GmILPA1 in vitro. Brackets denote the ubiquitinated bands. d, e Ubiquitination of GmGA2ox-like by GmILPA1 in N. benthamiana leaves via Agrobacterium-mediated transient expression. f GmGA2ox-like is ubiquitinated in plants. Ubiquitinated proteins were enriched from the P62-agarose matrix that was incubated with total proteins isolated from three independent transgenic plants stably expressing GmGA2ox-like-GFP or from H12 plants. g Regulation of GmGA2ox-like stability by GmILPA1 in vivo. H12 and Gmilpa1-2 seedlings grown under white light and white light supplemented with UV-B to the V2 stage, and GmGA2ox-like was checked with an anti-GmGA2ox-like antibody. The band intensities in western blots were quantified (h), Data are presented as mean values ± SEM, n = 3 independent experiments. i Immunoblots showing GmGA2ox-like protein levels in V2 stage seedlings of H12 and Gmilpa1-2 grown in white light (600 μmol m−2 s−1, 12 h/12 h light/dark) and then transferred to white light supplemented with UV-B (1.5 μmol m−2 s−1, 12 h/12 h light/dark) for the indicated time periods. GmGA2ox-like abundance was detected with anti-GmGA2ox-like antibody and quantified (k, l). Data are presented as mean values ± SEM, n = 3 independent experiments. j GmGA2ox-like degradation in cell-free degradation assays in the presence of GmILPA1 and GmUBL1. GmGA2ox-like-MYC, GmUBL1-GFP, and GmILPA1-FLAG were individually infiltrated in N. benthamiana. GmGA2ox-like-MYC protein extracts were individually mixed with recombinant GmUBL1-GFP, GmILPA1-FLAG, GmUBL1-GFP, or GmILPA1-FLAG. Samples were collected at the indicated time points, and GmGA2ox-like abundance was probed by immunoblotting with anti-MYC antibody and quantified (m). Data are presented as mean values ± SEM, n = 3 independent experiments. Source data are provided as a Source Data file.