Figure 5NEMO degradation assay. (A) HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence HA-tagged NEMO plasmid. NEMO degradation assay. (B) MARCH2+/+ and MARCH2-/- HEK293T cells were infected with PR8-GFP (MOI=1) in time-dependent manner. Cells were harvested following infection, NEMO expression level, total and phosphorylated IRF-3, and TBK-1 were measured by immunoblotting. β-actin was used to confirm equal loading of protein. C HEK293T cells transfected with a Strep-tagged empty vector or MARCH2 together with GST-tagged NEMO were treated with MG132 (10 μM), chloroquine, NH4Cl, or Z-VAD for 6 h before harvest. Whole cell lysates were subjected to immunoblotting with indicated antibodies. D HEK293T cells were transfected with different doses of Strep-tagged MARCH2 together with and GST-tagged NEMO plasmid. MG132 (10 μM) was added to cells for 6 h prior to harvest. Whole cell lysates were subjected to immunoblotting with the indicated antibodies. E Confocal microscopy was used to examine colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence or absence of MG132 (10 μM). Scale bar, 10μm. Arrow indicates the co-localized NEMO and MARCH2 protein. F HEK293T cells transfected with Strep-tagged empty vector or MARCH2 with GST-tagged NEMO and each different HA-tagged ubiquitin mutants (indicated lysine (K) only, other lysines (K) mutated to arginines (R) were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to pull down with GST beads, followed by immunoblotting with an anti-HA antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies. G NEMO ubiquitination assay. HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence of a HA-tagged NEMO plasmid. Lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-K48 antibody. MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). Viral replication was assessed under a fluorescence microscope (H) MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). by measuring GFP expression using a fluorescence modulator MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). (J) Levels of IFN-β (top) and IL-6 (bottom) in cell supernatants were measured in an ELISA. K In vitro ubiquitination assay for NEMO. HEK293T cells were transfected with GST-tagged NEMO, Strep-tagged MARCH2 or MARCH2CCH individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.