FIG 2.

Early ERK activation is dispensable for HPV16 infection. (A) Serum-starved HaCaT cells were either directly incubated with PBS (mock) or EGF for 10 min (left) or pretreated with 25 μM Iressa for 30 min and subsequently stimulated with EGF for 10 min (middle). To test for the reversibility of Iressa treatment, cells were preincubated with 25 μM Iressa for 30 min and stimulated with EGF after washout of the inhibitor for 10 min (right). Cell lysates were first blotted against pERK and thereafter against ERK. pERK levels were quantified and normalized to the mock of the untreated condition and ERK (loading control). (B) Serum-starved HaCaT cells were incubated with 25 μM Iressa for 30 min prior to binding of HPV16 PsVs or EGF for 1 h (mock = PBS). Subsequently, Iressa was washed out, and incubation was continued in the presence of HPV16 or EGF for the indicated time points. Lysates were processed as in panel A. pERK levels were quantified and normalized to the respective mock samples and ERK (loading control). (C) HaCaT cells were preincubated with 25 μM Iressa for 30 min and infected with HPV16 PsVs at different MOIs. At the indicated time points p.i., Iressa was washed out and infection was continued in the absence of the inhibitor. Cells were scored for infection 48 h p.i. by flow cytometry, and infection was normalized to the untreated control. Depicted is the mean of three experiments ± SD.