Supplementary Figure 3: Mass-spectrometry-based identification of novel linear polyUb-modified substrates.

(A) A schematic representation of the preliminary SILAC-based MS proteomic approach for the identification of linear polyUb-modified substrates upon 15 min of TNFα stimulation. Note that the SILAC labels were reversed in the subsequent triplicate MS analysis. (B) Scatter plots shown as a function of protein intensity and SILAC ratios (left: M/L, right: H/L) for preliminary MS data in (A) and Supplementary Table 1. SILAC ratios of log2>1 are marked in red. Several putative LUBAC substrates selected for the final validation are indicated in the plots. The number of identified protein pairs is marked in left upper part of each plot. (C) Experimental reproducibility of three independent experimental replicates of MS proteomic experiments from Figure 4a. The Pearson Correlation Coefficients are depicted in the left upper part of each plot. (D) Expression levels of putative linear polyUb-modified proteins in two different sets of T-REx HEK293T cell lines used in MS screens. Blot images for Supplementary Figure 3d were cropped to improve the conciseness of the presentation (full blots are available in Supplementary Figure 12).