Supplementary Figure 2: In vivo validation of INT-Ub.

(A) A comparison of INT-Ub (left panel), INT-Ub.7KR (middle panel), INT-Ub.ΔGG (right panel) and endogenous Ub levels in T-REx HEK293T cells upon doxycycline induction. To visualise levels of unconjugated Ub proteins, lysates were treated with USP2-cc. (B) A comparison of the effect of untagged Ub and Ub.ΔGG on NFκB transcriptional activity (control for Figure 2f). NFκB transcriptional activity was measured by a luciferase assay. Results are shown as means and s.e.m. (n=3). n.s=no statistically significant difference (p>0.05), determined by the two tailed Student’s t-test. (C) The effect of OTULIN C129A overexpression on the abundance of HMW linear polyubiquitinated proteins, was estimated by the linear Ub-specific Lub9 antibody upon denaturing INT-Ub.7KR Strep-Tactin PD. (D) The effect of TNFa stimulation (15 min) on the appearance of HMW ubiquitinated forms of NEMO upon denaturing INT-Ub.7KR Strep-Tactin PD. Blot images for Supplementary Figure 2a,c,d were cropped to improve the conciseness of the presentation (full blots are available in Supplementary Figure 12).