Loss of Wee1 protein affects only the isogenic RKO inactivated p53 cell line. (a) RKO-vector control (gray bars) and RKO-E6 (black bars) cell lines were subjected to 5 nM siRNA treatment for five days following plating. (b) RKO-vector control and RKO-E6 cell lines were subjected to 5 nM siRNA treatment for two days following plating. Direct cell lysates were analyzed by Western blotting to ascertain loss of Wee1 protein levels as well as the effects on its direct target (pCDK). p53 and p21 protein levels were assessed to determine effects of the HPV-E6 transgene. Total CDK1 and Actin were blotted to control for total protein levels.