Immunoblotting of Cell Signaling Proteins

Immunoblotting was performed as described previously (44 (link)). The following antibodies were used at 0.1 to 0.5 μg/ml: anti-pMCM2 (3378-1; Epitomics Inc.), anti-MCM2 (sc-9839; Santa Cruz Biotechnology), pChk1 (#2348; Cell Signaling Technology), anti-Chk1 (#2345; Cell Signaling Technology), anti-pCDK1 (#9111; Cell Signaling Technology), anti-CDK1 (sc-954; Santa Cruz Biotechnology), anti-pBRCA1 (#9009; Cell Signaling Technology), anti-pp53 (#9286; Cell Signaling Technology), anti-pATM (2152-1; Epitomics Inc.), anti-p21 (#2947; Cell Signaling Technology), anti-gH2AX (#2577; Cell Signaling Technology), anti-p53 (sc-126; Santa Cruz Biotechnology), anti-PARP1 (#9542; Cell Signaling Technology), anti-BRCA1 (sc-6954; Santa Cruz Biotechnology), anti-CDC7 (sc-56274; Santa Cruz Biotechnology), anti-DBF4 (ab124707; Abcam), and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (MAB374; Chemicon). Immunoblotted proteins were visualized using chemiluminescence.

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Iwai K., Nambu T., Kashima Y., Yu J., Eng K., Miyamoto K., Kakoi K., Gotou M., Takeuchi T., Kogame A., Sappal J., Murai S., Haeno H., Kageyama S.I., Kurasawa O., Niu H., Kannan K, & Ohashi A. (2021). A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery. Science Advances, 7(21), eabf0197.

Publication 2021

anti-MCM2 pChk1 anti-Chk1 anti-pCDK1 anti-CDK1 anti-pBRCA1 anti-pp53 anti-p21 anti-gH2AX anti-p53 anti-PARP1 anti-BRCA1 anti-DBF4 anti-GAPDH

Antibodies Brca1 Cdk1 Chemiluminescence Gapdh Glyceraldehyde 3 phosphate dehydrogenase Mcm2 Parp1 Proteins

Corresponding Organization : Takeda (United States)

Other organizations : National Cancer Center Hospital East

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Variable analysis

independent variables

Antibodies used for immunoblotting at 0.1 to 0.5 μg/ml: anti-pMCM2, anti-MCM2, pChk1, anti-Chk1, anti-pCDK1, anti-CDK1, anti-pBRCA1, anti-pp53, anti-pATM, anti-p21, anti-gH2AX, anti-p53, anti-PARP1, anti-BRCA1, anti-CDC7, anti-DBF4, anti-GAPDH

dependent variables

Immunoblotted proteins that were visualized using chemiluminescence

control variables

Experimental protocol was described previously in reference 44

controls

No positive or negative controls were explicitly mentioned in the information provided.

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