Fig. 4: COP1 mediates K63-linked ubiquitination of GH3.5 in the dark. | Nature Communications

Fig. 4: COP1 mediates K63-linked ubiquitination of GH3.5 in the dark.

From: Inactivation of GH3.5 by COP1-mediated K63-linked ubiquitination promotes seedling hypocotyl elongation

Fig. 4

a In vitro ubiquitination assay showing that COP1 ubiquitinates GH3.5. UBE1 (E1), His-UBC10 (E2), COP1-SPA4 complex (E3), and His-tagged ubiquitin were used for the assay. Ubiquitinated GST-GH3.5 was detected with anti-GST and anti-pan-Ub antibodies. b COP1-mediated ubiquitination of GH3.5 in Arabidopsis protoplasts. GH3.5-GFP plasmids alone or together with HF-COP1 plasmids were each transfected into protoplasts. Immunoprecipitated GH3.5-GFP proteins were used to detect the ubiquitination levels of the proteins. Actin was used as a loading control. c COP1-mediated ubiquitination of GH3.5 with the co-expression of the K63R mutant ubiquitin in protoplasts. MYC-Ub-WT, MYC-tagged wild-type ubiquitin. MYC-Ub-K63R, MYC-tagged ubiquitin with Lys63 substituted by arginine (R). Ubiquitination of immunoprecipitated GH3.5-GFP proteins was detected with an anti-MYC antibody. Levels (d) and linkage types (e) of ubiquitination of GH3.5-GFP in Col, GH3.5-GFP, and GH3.5-GFP/cop1-4 seedlings grown in the dark. GH3.5-GFP proteins were immunoprecipitated with GFP-trap magnetic beads, and their ubiquitination was detected with anti-pan-Ub, anti-K48 Ub and anti-K63 Ub antibodies. The asterisk in (d) indicates a non-specific band. HSP was used as a loading control. Source data are provided as a Source Data file.

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