Figure 2

Chk1-Ser³⁴⁵is exclusively required for Vpr-induced G2 arrest. HeLa cells were first transfected with wild type (WT) siRNA-resistant pEGFP-Chk1 (siR-Chk1) or pEGFP-Chk1 Ser345A mutant (siR-Chk1-S345A) plasmids. The endogenous Chk1 mRNA was then depleted by a specific Chk1 siRNA for 24 hrs followed by Adv or Adv-Vpr transduction. The symbol "+" indicates presence of the siR-Chk1 or siR-Chk1-S345A plasmids. The dash sign "-"means no plasmid was introduced in wild-type Chk1, depleted by siRNA. The cell cycle profiles of the indicated cell lines were measured 48 hours after the adenoviral transduction by flow cytometric analysis (A). Expression of endogenous or siRNA-resistant Chk1 constructs from indicated cell lines was confirmed by Western blot analysis using anti-Chk1 antibody at the same time of flow cytometric analysis (B). Note that the siR-Chk1 or siR-Chk1-Ser345A gene products cannot be depleted by the normal "Chk1 siRNA" used here because silent mutations were incorporated into the Chk1 genes during site-directed mutagenesis. These silent mutations will not alter the intended protein sequences, i.e., wild type Chk1 or Chk1-Ser345A.