Figure 5.

Transient overexpression of Mcl-1 increases phosphorylation of Chk1 at Ser345. (A) HeLa cells were transiently transfected with Mcl-1 and whole cell extracts immunoblotted for Mcl-1, phospho-Ser345 Chk1, or vinculin as a loading control. (B) Cells overexpressing Mcl-1, or empty vector, as in A, were analyzed for cell cycle status by flow cytometry. Increased percentage of cells in G2 after Mcl-1 expression showed a significant change (p = 0.05). (C) Hs68 primary fibroblasts were transiently transfected with empty vector (Con) or with Mcl-1 and extracts probed as in A, except for use of anti-Chk1 as a loading control. (D) HeLa cells were transiently transfected with either Mcl-1 or Mcl-1³⁰⁴ as indicated, and extracts were immunoblotted as in A, except for the additional anti-Chk1 blot, which also serves as a loading control. (E) HeLa cells were untreated (C) or treated with etoposide for 3 h (Et) or were transiently transfected with vector alone (V) or Mcl-1–containing vector (M). Chromatin extracts were prepared, separated, and immunoblotted for the presence of phosphorylated histone H2Ax (P-H2Ax) or for histone H4 (HH4) as a loading control. The pairs of samples separated by vertical lines were resolved on a single gel.