The N Terminus of Orf Virus-Encoded Protein 002 Inhibits Acetylation of NF-κB p65 by Preventing Ser276 Phosphorylation
Figure 7
ORFV002 interferes with NF-κB p65 (Ser276) phosphorylation induced by MSK1.
A. HEK293T cells were transfected using expression vectors for p65, Flag-MSK1 plus GFP, 002GFP, 002GM1 or 002GM3. Activation of MSK1 was achieved by co-transfecting MKK6 and p38 kinase. At 24 hours post-transfection, cells were treated with 20 ng/ml TNF-α for 30 minutes, and cell lysates were prepared and detected by immunoblotting with an anti-NF-κB-p65 (pSer276) antibody. B. HEK293T cells were co-transfected with plasmid p65 plus: lane 1: MKK6 + p38; lane 2: MKK6 + p38 + MSK1 + pEGFP; lane 3: MKK6 + p38 + MSK1 + 002GFP; lane 4: MKK6 + p38 + MSK1 + 002GM1; lane 5: MKK6 + p38 + MSK1 + 002GM2; lane 6: MKK6 + MSK1 + 002GM1; lane 7: p38 + MSK1 + 002GM2; lane 8: MKK6 + MSk1 + 002GM2; lane 9: MKK6 + p38 + MSK1 + 002GM3. At 24 hours post-transfection, cells were treated with 20 ng/ml TNF-α for 30 minutes, and cell lysates were prepared and detected by immunoblotting with an anti-NF-κB-p65 (pSer276) phosphorylation antibody antibody. C. The relative densitometry of NF-κB p65 (pSer276) phosphorylation bands normalized to the level for loading control (GAPDH). The results are representative of three independent experiments. NS: No significant difference.