Figure 3: Interaction of PAR and Chk1 in vivo is independent of Atr.
(a) Co-immunoprecipitation (IP) and non-denaturing slot-blot analysis of PAR, Chk1 and PARP1 interaction in pMEFs from indicated genotype and treatment. pMEFs without (−) or with 2 mM HU treatment (3 h) were IP and immunoblotted (IB) using antibodies against PAR, Chk1 or PARP1. Ten percent of the cell lysates were used as inputs. (b,c) Co-IP and non-denaturing slot-blot analysis of the interaction between PAR and Chk1 (b), or between PAR and p-Chk1 (c) in Atrf/f MEFs cultured without 4-OHT (−) or with 4-OHT (+, 2 μM). The MEFs, which were untreated (Untr) or treated with MNNG (2 μM for 3 h), H2O2 (10 μM for 30 min), 4 mM HU (3 h) or UV (100 J m−2), were IP and IB by antibodies against PAR, Chk1 or p-Chk1(S317). Ten percent of the cell lysates were used as Inputs. (d,e) Co-IP and non-denaturing slot-blot analysis of the interaction between PAR and Chk1 (d) or between PAR and p-Chk1 (e) in MEFs after DNA damage without (−) or with ATR inhibitor (ETP-46464, 10 μM) treatment for 2 h. The MEFs were untreated (Untr) or treated with MNNG (2 μM for 3 h), H2O2 (10 μM for 30 min), 4 mM HU (3 h) or UV (100 J m−2) and followed by IP and IB by the antibody against PAR, Chk1 or p-Chk1(S317), respectively. Ten percent of the cell lysates were used as input.
