Figure 3.

Inhibition of NF-κB activation by RVA NSP1s. (A–D) HEK-293 cells were transfected with a plasmid containing a firefly luciferase gene under the control of an NF-κB-responsive promoter, a plasmid directing constitutive expression of renilla luciferase as an internal control, either the empty vector pcDNA3.1, or pcDNA3.1 encoding the NSP1 protein from the indicated RVA strains, or MCV MC159 as a positive control. Reporter genes were activated by treatment with 5 ng/mL TNFα at 24 h post-transfection (A), or inclusion of a plasmid encoding IKKβ (B), β-TrCP (C) or p65 (D) in the transfection. 48 h after transfection, cells were lysed and levels of firefly and renilla luciferase were measured. The normalized activity of the reporter gene in the samples transfected with the empty vector and induced by TNFα (A), IKKβ (B), β-TrCP (C) or p65 (D) was set to 100%. Differences between induced samples containing NSP1 or MC159 and the induced control sample containing the empty vector were assessed using the Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).