Figure 4.

DNA damage-mediated phosphorylation of Chk1 at S345 is associated with p53-dependent checkpoint proteins. Phosphorylation of Chk1 on S345 in response to DNA damage and UCN-01 was measured by IP with antibody against S345p-Chk1 followed by an IB with Chk1 antibody (G4). The complexes of phosphorylated form of Chk1 with p53, 14-3-3σ, p21, and IgG were assayed by immunoblotting the membrane with anti-p53 monoclonal antibody (DO-1), anti-14-3-3σ goat polyclonal antibody (N-14), anti-p21 monoclonal antibody (WAF1), and anti-mouse IgG. The nonimmune serum was used as a control for the IP. Nonimmune serum did not precipitate Chk1, p53, 14-3-3σ or p21. (The amounts of extracts used for IPs were adjusted to have equal Chk1 levels in all lanes. This was confirmed by direct immunoblotting of whole-cell extracts with G4 anti-Chk1 and anti-mouse IgG antibodies, as shown in the lower bracketed section.)