Figure 5: The interaction of Chk1 with PAR and its canonical partners Claspin and BRCA1. | Nature Communications

Figure 5: The interaction of Chk1 with PAR and its canonical partners Claspin and BRCA1.

From: Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

Figure 5

(a) Co-IP and non-denaturing slot-blot analysis of PAR, Chk1 and PARP1 interaction in U2OS cells transfected with mock (Non), Flag-tagged empty vector (Flag-EV), wild-type Chk1 (Flag-wtChk1), and PbR-mutant Chk1 (Flag-PbRC/AChk1) and treated with or without HU (2 mM) for 3 h. Cell extracts at 0 h after HU removal were IP and IB by antibodies against Flag (for Chk1), PAR or PARP1. Ten percent of the cell lysates were used as an input. (b–e) The PbR mutation does not interfere with the interaction of Chk1 with Claspin or BRCA1. (b,d) Co-IP of Chk1-Claspin and Chk1-BRCA1. HEK293 cells were transfected with HA-Chk1 and Flag-Claspin (b), or Flag-Chk1 and HA-BRCA1 (d) and treated with or without HU (4 mM) for 3 h. Cell extracts at 0 h after HU removal were IP and IB by the antibodies against Flag- and HA-tag as indicated. (c,e) Quantifications of the signal intensity of the indicated proteins after normalization by respective loading (IB with HA or Flag in lower panels of each IPs) from three samples of two independent experiments. NS, not significant. Error bars represent the s.e.m.

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