HPV16 activates Cdc42 protein at specific timepoints after viral addition. Active Cdc42 was evaluated with a pull-down using PAK-PBD coated beads. (A) Western blot of samples treated with (right side) or without (left side) virus for a single experiment. Blots for Cdc42 total protein, active Cdc42 (GTP bound), and actin. Levels of active Cdc42 was determined for cells incubated with virus for 5 min, 15 min, 30 min, and 60 min. (B) Positive control showing Cdc42 activation induced by 200 ng/mL EGF for 15 min. (C) Validity of PAK-PBD coated beads were evaluated with control lysates treated with GTPỿ and GDP for 15 min. (D) Densitometry data for one individual activation pulldown of Cdc42 with PAK-PBD glutathione beads represented in A. Relative active Cdc42 protein was normalized to 0-min timepoint. A peak in Cdc42 activity after 15 min of viral addition. (E) Mean values of seven separate activation pulldown experiments for GTP bound Cdc42. Activation of Cdc42 after viral addition occurs most often between 15–30 min.