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. 2022 Jan 28;34(4):1354–1374. doi: 10.1093/plcell/koac014

Figure 9.

Figure 9

Overexpressing ATL31 enhances low C/high N-nutrient-responsive ubiquitination of SYP61. Immunoblot analysis to examine the effect of ATL31 on SYP61 ubiquitination in low C/high N-nutrient condition. Arabidopsis plants expressing mRFP-SYP61 (53 kDa; pSYP61:mRFP-SYP61) in the WT or ATL31-GFP-overexpressing background (2xp35S:ATL31-GFP) were grown for 11 days in liquid medium containing 100-mM glucose/30-mM nitrogen and treated with medium containing 0-mM glucose/60-mM nitrogen for the indicated time. WT and 2xp35S:ATL31-GFP plants were used as negative controls. Extracted proteins were immunoprecipitated with anti-RFP antibody beads and detected with anti-mCherry, anti-ubiquitin (FK2), and anti-K63-linked ubiquitin (Apu3) antibodies. ATL31-GFP in the crude extract was detected with anti-GFP antibody. Arrowheads indicate ubiquitinated mRFP-SYP61. The numbers represent the relative intensities of the di-Ub bands (the bands indicated by the lower arrowhead in the anti-ubiquitin blot) normalized by the band intensity of mRFP-SYP61. Glc, glucose; N, nitrogen; unit, mM.