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. 2012 Sep 28;7(9):e46515. doi: 10.1371/journal.pone.0046515

Figure 5. The effect of A20 on CVB3-induced NF-κB signaling in cardiac myocytes.

Figure 5

Cardiac myocytes were pre-infected with adenovirus (Ad-LacZ or Ad-A20) to over-express A20 or lentivirus (LV-ctrl or LV-shA20) to knock down endogenous A20 for 48 h, then exposed to CVB3 (MOI = 10) for the indicated time. (A and D) Cell lysates were examined for phosphorylation of IκB-α, p65 and protein expression of total IκB-α, p65 and A20. β-actin was probed as the loading control. Left, representative western blots; Right, quantitative results, the band intensity was measured and the ratio of phospho-IκBα and phospho-p65 to β-actin was calculated. (B and E) NF-κB p65 DNA binding activity of nuclear extracts from cardiac myocytes was measured using the NF-κB p65 transcription factor assay kit. (C and F) Cell lysates were harvested at the indicated time, immunoprecipitated with anti-IKKγ antibody, incubated with GST-IκBα substrate, and analyzed by immunoblotting for phospho-GST-IκBα levels. Left, representative western blots; Right, quantitative results. Each assay was done 3 times. Values were presented as the means±SEM. *, P<0.05; **, P<0.01; ***, P<0.001.