Supporting Figures

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Fig. 7. TNF-a activation of NF-kB is impaired in response to CVB3. HeLa cells were transfected with a WT kB element-luciferase reporter construct, and then infected with CVB3 for 30 min or treated with TNF-a for 30 min, or infected with CVB3 and treated with TNF-a for 30 min. (Cells were also cotransfected with a construct consisting of a CMV promoter fused to a Renilla luciferase reporter as an internal standard.) Luciferase expression was measured by a luminometer (n = 3 ± SD; *, P < 0.05).

Fig. 8. A partially inactive 3C^pro mutant does not cleave IkBa in vitro. Recombinant GST- IkBa was incubated with recombinant 3C^pro(C147S) mutant, fractionated by SDS/PAGE, and immunoblotted with antibody to the N terminus of IkBa.

Fig. 9. A partially inactive 3C^pro mutant does not retain NF-k B in the nucleus. HeLa cells were transfected with a plasmid expressing a partially inactive 3C^pro(C147S) mutant. Transfected cells were washed with PBS, preincubated 90 min in serum-free medium with 100 mg/ml cyclohexamide (CHX), and then treated for 30 min with 10 ng/ml TNF-a. plus 100 mg/ml CHX (CHX/TNF). The cells were then washed and subsequently incubated for 90 min in the absence of serum. Cells were fixed, permeabilized, and assayed for p65 (FITC, green).