Journal: Nature Communications

Article Title: The ribosome-associated N-terminal acetyltransferase B coordinates global proteostasis and autophagy in plants by creating Ac/N-degrons

doi: 10.1038/s41467-026-71208-2

Figure Lengend Snippet: a , b Immunoblot detection and quantization of K63-type poly-ubiquitination (red line) after enrichment with the Ubi-Qapture matrix from wild type (WT) and naa20-cr1 treated with 1 μM ConA for 4 h. The K63-ubiquitination levels were normalized to the input and set to 1 in WT. Data represent the means ± SEM ( n = 3 independent replicates). c – e Immunoblot detection of ATG8 ( c ), ATG3 ( d ) and ATG5-ATG12 complex ( e ) from wild type (WT) and natb mutants. In ( c , e ), atg5-1 mutant was used as a control for antibody specificity. Data represent the means ± SEM ( n = 4 biologically independent samples). Protein levels were normalized to the loading control (LC) and the detected signal was set to 1 in WT. f The transcript levels of ATG genes in wild type (WT) and naa20-cr1 determined by qRT-PCR. Actin7 ( At5g09810 ) served as the reference gene. Data represent the means ± SEM ( n = 3 for ATG8e or 4 for ATG8a/3/5 biologically independent samples). g Confocal microscopy analysis of the autophagic bodies in cells of the root elongation zones of five-day-old pUBQ:GFP-ATG8a/WT and pUBQ:GFP-ATG8a/naa20-cr1 seedlings after incubation with 1 μM ConA in the dark or with DMSO under the light for six hours. A representative picture from 10 individual seedlings is shown. Scale bar, 50 μm. Inset scale bars, 10 μm. h Quantitative analysis of the number of detected autophagic bodies per frame in ( g ). Data represent means ± SEM from 10 images taken from individual roots. Different letters indicate individual groups identified by pairwise multiple comparisons with a one-way ANOVA followed by a Tukey’s test (p < 0.05). i GFP-ATG8a cleavage assay showing a higher GFP/GFP-ATG8a ratio in the five-day-old naa20-cr1 background seedlings upon carbon-starvation treatment. The values below the immunoblot are the relative ratios of free GFP to GFP-ATG8a. In ( a , c – e , i ), amido black staining of proteins transferred to the PVDF membrane served as LC and all experiments were repeated three times with consistent results. In ( b , f ), significance was determined by the two-sided Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Enrichment of all type- and K48-polyubiquitinated proteins was performed using Ub i Qapture-Q matrix kit (Enzo life Sciences) as previously described in ref. . Proteasome and deubiquitination activity were inhibited in the leaves of wild type and natb plants by floating leaf discs on 1⁄2 Hoagland medium supplemented with 50 μM of MG132 (Santa-Cruz Biotechnology) and 20 μM deubiquitinase inhibitor PR-619 (Sigma Aldrich) for 6 h. To quantify the K63-polyubiquitinated proteins, 1 μM ConA (Santa-Cruz Biotechnology) instead of 50 μM MG132 was applied to the 1⁄2 Hoagland medium to inhibit autophagy pathway.

Techniques: Western Blot, Ubiquitin Proteomics, Mutagenesis, Control, Quantitative RT-PCR, Confocal Microscopy, Incubation, Cleavage Assay, Staining, Membrane