Figure 2.
The phosphorylation of Atr substrates are diminished in Bid-deficient cells following replicative stress. (a) Phosphorylated mouse Chk1 presents as a shifted band. Bid+/+ and Bid−/− MPCs were treated with 10 mM HU for 2 h. Whole-cell extracts were incubated with 10 U Calf Intestinal Alkaline Phosphatase (Invitrogen)/100 μg lysate, resolved by SDS-PAGE and immunoblotted with anti-Chk1, anti-pChk1 (S317), or anti-pChk1 (S345) as indicated. Solid arrows denote the mobility of shifted phosphorylated Chk1, and dashed arrows denote the mobility of unphosphorylated Chk1. (b) Bid+/+ and Bid−/− MPCs were treated 10 mM HU for the indicated times. Total cell lysate was resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. (c) U2OS cells were treated with Bid-specific siRNA no.7, Bid-specific siRNA no.8, or control siRNA for 72 h. Bid KD and control KD cells were treated with 10 mM HU or 25 μM ETOP for 2 h, and total cell lysate was resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. (d) U2OS cells transfected with control siRNA or Bid siRNA (no. 8) for 72 h were treated with 10 mM HU for 2 h. Whole-cell lysates were immunoprecipitated with anti-Chk1 antibody, and the immunoprecipitated product was incubated with 1 μg GST-Cdc25C protein, 10 μM cold ATP and 5 μCi γ-32P-ATP in kinase buffer. Chk1 kinase reactions were resolved on SDS-PAGE, stained with SimplyBlue SafeStain (Invitrogen) to visualize GST-cdc25c levels, and analyzed by autoradiography. (e) Bid+/+ and Bid−/− MPCs were treated 10 mM HU over time. Total cell lysate was resolved by SDS-PAGE followed by immunoblot with the indicated antibodies. Relative band intensity has been measured by densitometry analysis. (f) U2OS cells were transfected with control siRNA or Bid siRNA for 72 h, and then treated with 10 mM HU over time. Total cell lysate was resolved by SDS-PAGE followed by immunoblot with the indicated antibodies. Solid arrow denotes the mobility of shifted phosphorylated RPA32, and dashed arrow denotes the mobility of unphosphorylated RPA32. Relative band intensity was measured by densitometry analysis