Figure 7.
The I226R protein promotes the degradation of NEMO through enhancing its ubiquitination: (A–C) 293T cells were co-transfected with 1 μg I226R-Flag or EV and 1 μg IKKα-HA (A), IKKβ-Flag (B), or NEMO-Flag plasmids (C). Western blotting was then performed. (D) 1 μg NEMO-Flag was co-transfected with varying amounts of I226R-Flag plasmid in 293T cells. Western blotting was then performed. (E) 293T cells were transfected with 3 μg I226R-Flag or EV and infected with SeV for 12 h. RT-qPCR was performed to detect the mRNA levels of NEMO. (F) 1 μg NEMO-Flag was co-transfected with 1 μg I226R-Myc or EV and subjected to 50 μg /mL CHX treatment for indicated hours. Then, Western blotting was performed. (G,H) 293T cells were co-transfected with 1 μg I226R and 1 μg NEMO and treated with 50 μg/mL CHX and either 20 mM MG132, 50 mM CQ, 20 mM NH4Cl, or DMSO. Western blotting was performed to evaluate the expression of NEMO. Relative levels of NEMO were quantitated by densitometry and normalized to β-actin levels (H). (I) 293T cells were transfected with 1.5 μg I226R-Myc or EV, along with 1 μg NEMO-Flag and 1.5 μg Ub-HA. At 24 h after transfection, IP and Western blotting were performed to evaluate the ubiquitination levels of NEMO. Statistical analysis was performed using the Student’s t-test. * p < 0.05; ** p < 0.01.