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. 2024 May 21;7(8):e202402740. doi: 10.26508/lsa.202402740

Figure 3. Ubiquitin chain length–dependent interactors.

Figure 3.

(A, B) Correlation of homotypic Ub chain interactomes within (A) chloroacetamide (CAA) and (B) N-ethylmaleimide (NEM) datasets. The Spearman correlation was calculated using the moderated F values for each Ub pulldown of significant differently enriched interactors. Significant differently enriched interactors were identified by a moderated F test of prefiltered Ub-enriched proteins (Adj.P < 0.05). (C, D) Scatterplot of Ub3 versus Ub2 interactor enrichment. Comparison of K48 Ub3 versus K48 Ub2 in (C) and K63 Ub3 versus K63 Ub2 in (D), by a two-sample moderated t test. Comparison from CAA data plotted on the y-axis and from the NEM dataset plotted on the x-axis. Dot colours indicate significance: orange is statistically significant in both datasets, pink is significant in the CAA dataset only, teal blue is significant in the NEM dataset only, and grey is not significant in either dataset. Labelled proteins were significant in at least one dataset and have consistent fold change directions in both datasets: log(FC) < −0.5 or > 0.5 for K48, and log(FC) < 0 or > 0 for K63 (as indicated by the dotted line arrows). DDI2 is labelled despite log(FC) = 0.39 with NEM because of interest based on the same length preference for yeast homologue Ddi1. Adj.P < 0.05. (E) Heatmap based on MS data and Western blot validation of length-dependent enrichment of selected interactors. DDI2 enriched from the CAA-treated lysate (left) and APPL1 enriched from the NEM-treated lysate (right). Heatmap using moderated F values (moderated F test of prefiltered Ub-enriched proteins, Adj.P < 0.05). The same Ub interactor pulldown experiment as in Fig 1B, and thus the same silver stain of input Ub. Pulldown blotted using anti-APPL1 and anti-DD2 antibodies. (F) Table of identified Ub chain length–dependent interactors. Linkage-type preference was identified from a two-sample moderated t test of K48 Ub3 versus K63 Ub3. Adj.P < 0.05. (F) Chain length preference validated by Western blot, (F) or (Fig S11), or literature search.