Fig 2: Nedd4l-catalyzed K29-linked ubiquitination of TRAF3.a IB of total ubiquitination, K48- and K63-linked ubiquitination of TRAF3 in VSV-infected wild-type (Nedd4l+/+) and Nedd4l-deficient (Nedd4l-/-) peritoneal macrophages. b Plasmids expressing Flag-TRAF3, HA-Ub, and Myc-Nedd4l were co-transfected in 293T cells. After 24 h, cells were infected with VSV (MOI = 10) for 12 h. IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 with indicated antibodies. c IB analysis of TRAF3 incubated with Nedd4l recombinant proteins and ubiquitination reaction components in vitro. d IB analysis of total ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, HA-Ub, and Myc-tagged wild-type Nedd4l or mutant Nedd4l with indicated antibodies. e IB analysis of ubiquitination of Flag-TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, Myc-Nedd4l, and HA-tagged wild-type Ub or HA-tagged mutant Ub (K11R, K29R, K33R, K48R, and K63R) with indicated antibodies. f IB analysis of ubiquitination of Flag-TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, Myc-Nedd4l, and HA-tagged wild-type Ub or HA-tagged mutant Ub (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only, Ub-K null) with indicated antibodies. Results in (a–f) are representative of three independent experiments.

Fig 3: Nedd4l promotes TRAF3 to associate with cIAP1/2 and HECTD3.a IB of Flag-cIAP1, Flag-cIAP2, Myc-Nedd4l, and HA-TRAF3 co-immunoprecipitated with anti-HA from lysates of 293T cells transfected with indicated plasmids. b IB of Flag-HECTD3, Myc-Nedd4l and HA-TRAF3 co-immunoprecipitated with anti-HA from lysates of 293T cells transfected with indicated plasmids. c IB of Flag-cIAP1, Flag-TRAF3, and Flag-TRAF3(C56R) co-immunoprecipitated with anti-TRAF3 from lysates of 293T cells transfected with indicated plasmids. d IB of Flag-HECTD3, Flag-TRAF3 and Flag-TRAF3(C56R) co-immunoprecipitated with anti-TRAF3 from lysates of 293T cells transfected with indicated plasmids. e RT-qPCR analysis of IFN-ß, IL-6, and TNF-a mRNA expression in 293 cells transfected with plasmids expressing TRAF3 or mutant TRAF3 and then infected with VSV (MOI = 10) for 12 h. Data are presented as mean ± SD (n = 3 per group) and p-values by two-tailed unpaired Student’s t-test are indicated in e. Results in (a–e) are representative of three independent experiments.

Fig 4: Nedd4l promotes TRAF3-dependent signaling.a Peritoneal macrophages from wild-type (Nedd4l+/+) and Nedd4l-deficient (Nedd4l-/-) mice were infected with VSV (MOI = 10) for indicated time. Phosphorylated and total TBK1, IRF3, p38, JNK, ERK1/2, NF-kB p65, and I?Ba were detected by western blot. Intensities of p-TBK1, TBK1, p-IRF3, and IRF3 signals in the three independent experiments were quantified by ImageJ and averages of the signals were shown in graphs. b Wild-type (Nedd4l+/+) and Nedd4l-deficient (Nedd4l-/-) peritoneal macrophages were infected with VSV (MOI = 10) for indicated time, followed by immunoprecipitation (IP) with anti-TRAF3 and immunoblot (IB) analysis with antibodies specific for TBK1, cIAP1/2, HECTD3, TRAF3, Nedd4l, and GAPDH (WCL: whole-cell lysates). c IB of Myc-TBK1, Flag-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Flag from lysates of 293T cells transfected with indicated plasmids. d IB of HA-MAVS, Flag-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Flag from lysates of 293T cells transfected with indicated plasmids. Results in (a–d) are representative of three independent experiments.

Fig 5: Nedd4l interacts with TRAF3.a IB of HA-MAVS, HA-TBK1, HA-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Myc from lysates of 293T cells transfected with indicated plasmids with HA-tag, Myc-tag, or GAPDH antibodies. b IB of TRAF3 and Nedd4l co-immunoprecipitated with anti-TRAF3 from lysates of C57BL/6 mice peritoneal macrophages infected with VSV (MOI = 10) for indicated time. c In vitro GST-pull-down assay of Nedd4l with GST or GST-fused TRAF3. d IB of TRAF3 and Nedd4l co-immunoprecipitated with Myc-tag-specific antibody from lysates of 293T cells co-transfected with plasmids expressing Flag-TRAF3 and Myc-tagged wild-type Nedd4l or mutant Nedd4l with CW (?CW),C2 (?C2), or HECT (?HECT) domain deleted. Results in (a–d) are representative of three independent experiments.