Figure 10.

Selective reduction in MAGI-1 protein levels in vivo by high-risk HPV E6 proteins. (a) Decrease in the steady-state protein levels of MAGI-1 induced by high-risk HPV E6 oncoproteins. Extracts of RIPA buffer-lysed COS-7 cells transfected with 0.5 μg of GW1-HAMAGI-1c plasmid alone or in combination with 4 μg of a GW1 plasmid expressing the indicated wild-type or mutant HPV E6 protein were immunoblotted with HA antibodies. (b) Failure of 18E6 to bind a MAGI-1 deletion mutant missing all five PDZ domains (MAGI-1Δ5PDZ) or the wild-type ZO-2 protein. Extracts of RIPA buffer-lysed COS-7 cells transfected with 5 μg of the indicated GW1 expression plasmid were subjected to GST-pulldown reactions with either GST or GST-18E6 protein. Recovered proteins were immunoblotted with HA antibodies. COS-7 extracts representing one-tenth the amount used in GST pulldown reactions were also directly immunoblotted with HA antibodies as a control. (c) Inability of 18E6 to reduce the steady-state protein levels of MAGI-1Δ5PDZ or ZO-2 in cells. Extracts of RIPA buffer-lysed COS-7 cells transfected with 0.1 μg of GWI-HAMAGIΔ5PDZ plasmid (upper panel) or with 0.01 μg of GW1-HAZO-2 plasmid (lower panel), alone or in combination with 4 μg of GW1 plasmid expressing wild-type or mutant HA18E6, were immunoblotted with HA antibodies