Figure 4(A)Immunoblot analysis of NEDD4L, MEKK2, and MEKK3 immunoprecipitated with NEDD4L-specific antibody from lysates of HeLa cells pre-treated with 20μM MG-132 for 6 h and then treated with IL-17 for the indicated time. Data information: All experiments were performed three times, and representative data are shown.(B)Immunoblot analysis of NEDD4L precipitated with GST-tagged Null or GST-tagged MEKK2 recombinant protein. Data information: All experiments were performed three times, and representative data are shown.(C)Immunoblot of HA-tagged MEKK2 co-immunoprecipitated with anti-Myc antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted. Data information: All experiments were performed three times, and representative data are shown.(D)Immunoblot analysis of MEKK2 and NEDD4L in Nedd4l+/+, and Neddl4- /- treated with DMSO, 20μM MG-132, or 0.2 μM bafilomycin A1 (Baf A1) for 6 h. Densitometry of MEKK2 was relative to actin. Data information: All experiments were performed three times, and representative data are shown.(E)Immunoblot analysis of MEKK2 incubated with NEDD4L or TRAF6 recombinant protein and the full complement of ubiquitination reaction components in vitro. Data information: All experiments were performed three times, and representative data are shown.(F)Immunoblot analysis of K48-, K63-linked, and total ubiquitination of MEKK2 following precipitation of MEKK2 with MEKK2-specific antibody from lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with MG-132. Data information: All experiments were performed three times, and representative data are shown.(G)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following precipitation of MEKK2 with Flag-tagged specific antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted. Data information: All experiments were performed three times, and representative data are shown.(H)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6O, K11O, K27O, K29O, K33O, K48O, K63O mutant Ub. (I)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6R, K11R, K27R, K29R, K33R, K48R, K63R mutant Ub. Data information: All experiments were performed three times, and representative data are shown.(J)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, increased dosage of Myc-tagged NEDD4L, and HA-tagged wild-type, K27O, or K63O mutant Ub. Data information: All experiments were performed three times, and representative data are shown.