Figure 3A. Western blot analysis of anti-FLAG (ZBP1) immunoprecipitation from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h. Blots are representative of three biological replicates. Immunoprecipitated material was treated or not with USP21 (1 μM) for deubiquitination. Input loaded was 5% for ZBP1 and 1% for co-immunoprecipitants. Asterisk indicates antibody heavy chain signal in IP samples.B. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination status of RIPK1, ZBP1 and RIPK3 in HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16h. Blots are representative of at least three biological replicates. After enrichment, samples were treated or not with USP21 (1 μM) for deubiquitination. Asterisk indicates unspecific bands. Arrows indicate RIPK3 signal.C. Enrichment of Ub-conjugates for analysis of the ubiquitination status of RIPK1 and ZBP1 using GST-1xUBA or linkage-specific SUBs in HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h.D. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination status of RIPK1 and ZBP1 in HT29, HT29 / TO-ZBP1WT and HT29 / RIPK1-KO aA3 / TO-ZBP1WT cells treated with 500 ng/mL Dox for 16 h. Blots are representative of four biological replicates. Asterisk indicates antibody background signal.E. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and GFP (Ctrl) or GFP-tagged SUBs as indicated. Luciferase reporter activities were measured 24 h after transfection and normalized to GFP+EV-transfected condition. Data is plotted as mean with S.E.M. (n = 4 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between the indicated condition and ZBP1/GFP-transfected condition. **** p < 0.0001. Cell lysates were analyzed by Western blotting to determine expression of ZBP1, GFP or GFP-tagged SUBs and Actin. Blots are representative of three biological replicates.F, NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and variants of OTULIN. Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1/EV-transfected cells. Data is plotted as mean with S.E.M. Multiple Welch t-tests were used to test for statistical differences between the indicated condition and the ZBP1/EV-transfected condition. (F) n = 4 biological replicates. ** p = 0.0012, ns = not significant (p = 0.2342). Cell lysates were analyzed by Western blotting to determine expression of ZBP1, or OTULIN, and Actin. Blots are representative of (F) four biological replicates.G. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and variants of CYLD Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1/EV-transfected cells. Data is plotted as mean with S.E.M. Multiple Welch t-tests were used to test for statistical differences between the indicated condition and the ZBP1/EV-transfected condition. (G) n = 3 biological replicates. * p = 0.0137; ns = not significant (p = 0.8374). Cell lysates were analyzed by Western blotting to determine expression of ZBP1, CYLD and Actin. Blots are representative of (G) three biological replicates.