Supplemental Figures

This article contains the following supporting material:

• Supplemental Figure 1 - Activation of Chk1 kinase after aphidicolin treatment. A549 cells expressing ATRIP-Myc or ATRIP-LG-Myc were lysed, and the lysates were subjected to immunoprecipitation using the anti-Chk1 antibody, and the immunoprecipitates were used for immunoblotting (upper) or the Chk1 kinase assay (lower). Relative Chk1 activities were examined using the K-LISA Checkpoint activity kit (Calbiochem). The data are the averages ± S.D. of three independent experiments.
• Supplemental Figure 2 - Knockdown of endogenous ATRIP by siRNA and complementation by siRNA-resistant ATRIP. (A) Knockdown of endogenous ATRIP by ATRIP siRNA. A549 cells were infected with retrovirus vector expressing ATRIP-Myc or ATRIP-LG-Myc that was immune to the ATRIP siRNA by mutations of wobble base pairs in the target sequence (see Materials and Methods). Then, the infected cells were infected with retrovirus expressing shRNA against ATRIP. Cell lysates were analyzed with immunoblotting using the anti-ATRIP antibody. (B) Modification of target proteins of ATR in the endogenous ATRIP-repressed cells. Cells expressing ATRIP shRNA used in (A) were untreated or treated with aphidicolin for 24 h (upper), or were untreated or treated with IR (lower). Cell lysates were subjected to immunoblotting as in Figure 5. (C) Spindle formation after aphidicolin treatment. The A549 cells were infected with retrovirus harboring the RNAi-resistant (RNAi-R) version of ATRIP-Myc or ATRIP-LG-Myc as in (A). The cells were untreated or treated with siRNA against ATRIP to repress the endogenous ATRIP, followed by treatment with aphidicolin for 24 h, and were examined as in Figure 7B.