Human Papillomavirus (HPV) Upregulates the Cellular Deubiquitinase UCHL1 to Suppress the Keratinocyte's Innate Immune Response
Figure 6
UCHL1 reduces phosphorylation levels of IRF3 and p65 and degrades NEMO in hrHPV-positive KC.
(A) UCHL1 knock down effect on poly(I∶C) stimulated p65 phosphorylation in HPV16+ keratinocytes. Monolayer cultures of shControl or shUCHL1-expressing HPV16+ KCs were stimulated for 0, 3 or 24 hours with Poly(I∶C). Whole cell extracts were analyzed by western blotting for p65, p65-p and β-Actin (as loading control). The relative expression of p65-p was quantified by measuring its density and by normalizing it to that of β-Actin. The expression levels of p65-p in the 0 h Poly(I∶C) cells were set to 100% for both shControl and shUCHL1 cells. The p65-p levels in the 3 h and 24 h Poly(I∶C) cells were calculated against the levels measured at 0 h Poly(I∶C) (right panel). (B) NEMO protein levels after knock down of UCHL1 in HPV16+ KCs. Monolayer cultures of shControl or shUCHL1-expressing HPV16+ KCs were treated with 100 µM cycloheximide (CHX) for 16 hours. Whole cell extracts were analyzed by western blot using antibodies against NEMO and β-Actin (control for protein content). The relative expression of NEMO was quantified by measuring its density and by normalizing it to that of β-Actin. The expression of NEMO in the DMSO control was set to 100% (right panel). (C) UCHL1 knock down effect on poly(I∶C) stimulated IRF3 phosphorylation in HPV16+ keratinocytes. Similar to A, however cell extracts were analyzed by western blotting using antibodies against IRF3, IRF3-p and β-Actin (as loading control). The relative expression of IRF3-p was quantified by measuring its density and by normalizing it to that of β-Actin. The expression of IRF3-p in the 3 h Poly(I∶C) control cells (no knock down of UCHL1) was set to 100% (right panel).