And-1 antibody was obtained from Novus (#NBP1-89091, dilution: 1:500 for WB and 1:100 for IF). BRCA1 (D-9, dilution: 1:100 for IF, Santa Cruz) and (C-20, 1:200 for WB and IP, Santa Cruz), HA (H9658, dilution: 1:1000 for WB and IP, Sigma), FLAG (F3165, dilution: 1:1000 for WB and IP, Sigma), γH2AX (05-636, dilution: 1:500 for IF, Millipore), CtIP (61141, dilution: 1:500, Active Motif) for IF and (sc-28324, dilution 1:11 000) for WB, Chk1 (sc-7898, dilution: 1:1000 for WB, Santa Cruz), and P-Chk1-317 (2344S, 1:500 for WB, Cell Signaling), MDC1 (ab11171, 1:500 for IF), NBS1(ab32074, 1:200 for IF) were used. Antibodies against RPA, RAD51 and 53BP1 were previously described (47 (link)–49 (link)). And-1 full-length was subcloned from pEFF-And-1(a kind gift from Dr Zhu) (44 (link)) into the FLAG-tagged vector (pIRES2-EGFP) and HA-tagged vector (pCMV-HA) and GFP-tagged vector (pFUGW). And-1 deletion mutants were generated by site-directed mutagenesis and confirmed by sequencing. Full-length CtIP and D1–D6 deletion mutants were kindly provided by Dr Chen (50 (link)).
Protocol detail
Comprehensive Immunostaining Protocol for DNA Damage Response
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Corresponding Organization :
Other organizations : Beijing Proteome Research Center, Shenzhen University, Tianjin Medical University, China Three Gorges University, Tianjin University of Science and Technology
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Variable analysis
independent variables
- And-1 antibody dilution
- BRCA1 antibody dilution
- HA antibody dilution
- FLAG antibody dilution
- γH2AX antibody dilution
- CtIP antibody dilution
- Chk1 antibody dilution
- P-Chk1-317 antibody dilution
- MDC1 antibody dilution
- NBS1 antibody dilution
- And-1 full-length and deletion mutants
- CtIP full-length and deletion mutants
- Protein expression/localization (assessed by Western blot and immunofluorescence)
- Not explicitly mentioned
- Antibodies against RPA, RAD51 and 53BP1 were previously described
- Not explicitly mentioned
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