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Fig. 4 | Genome Biology

Fig. 4

From: An ORFeome of rice E3 ubiquitin ligases for global analysis of the ubiquitination interactome

Fig. 4

Identification of BTB-type E3 ligases that interact with NRR and rTGA2.1. a Confirmation of the interaction between NRR and seven candidate E3 ligases. OsNPR2 and OsNPR3 (labeled in blue) were identified in a previous study. DDO, SD-Leu-Trp, QDO, SD-Leu-Trp-His-Ade. b Confirmation of the interaction between rTGA2.1 and five candidate E3 ligases. OsNPR2 and OsNPR3 (labeled in blue) were identified in a previous study. DDO, SD-Leu-Trp, QDO, SD-Leu-Trp-His-Ade. c Interaction network among NRR and rTGA2.1 and 11 candidate E3 ligases. The pink color line represents the identified interaction using the UbE3 library, and the blue color line represents the literature-reported interaction. d, e Co-IP assay of NRR or rTGA2.1 with their candidate E3 ligases in vivo. N. benthamiana leaves were agro-infiltrated with the indicated plasmid combinations. Total protein was extracted and immunoprecipitated with anti-HA/Agarose beads. Immunoblot detection was performed using anti-GFP or anti-HA antibody as indicated. f, g Degradation assay of NRR and rTGA2.1 by their candidate E3 ligases with or without 50 μM MG132 treatment. Plasmids harboring NRR-GFP (f) or rTGA2.1-GFP (g) and with their respective candidate E3 ligase genes fused with HA tag were co-expressed in rice protoplasts. GUS-HA was used as a negative control. At 16 h after co-transfection, 50 μM MG132 or an equal volume of DMSO solution was added to the protoplasts. The protoplasts were collected for protein extraction 4 h after MG132 treatment. NRR and rTGA2.1 protein abundance was detected by immunoblotting using an anti-GFP antibody, and E3 ligases were detected by an anti-HA antibody. HSP was used as an internal control

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