pChk1 levels are elevated prior to the onset of TMZ-induced DNA DSB, Chk2 activation, and cell cycle arrest.

<p>Isogenically paired U87 cells differing only in MGMT expression (U87 or U87+MGMT)(A- C) or G55 cells expressing MGMT or depleted of MGMT by pre-incubation with the MGMT-depleting agent BG (20 µM, 2 hours, G55+BG)(C- E) were incubated with vehicle (U) or TMZ (100 µmol/L, 3 hours) after which TMZ was removed, vehicle or BG was replaced, and cells were harvested at 24–72 hours (A, D) or at earlier time points (1–24 hours, B, C, E) following TMZ exposure for analysis of pChk1 (ser345), Chk1, pChk2 (thr68), Chk2, MGMT, pcdc25C (ser216), cdc25C, and β-actin expression by Western blot. For panel C, an ATR (or control IgG) immunoprecipitate was first prepared and incubated with the ATR substrate PHAS, after which a Western blot analysis of the extent of ATR-mediated PHAS ser/thr phosphorylation was performed. Mean fold induction of protein expression was based on densitometric measurements and is shown (relative to untreated controls) below the relevant immunoreactive bands. *, p<.05.