Nucleated Ter119+ sorted cells from wild-type and Parp-2−/− mice were washed once with PBS, and total protein extracts were prepared by directly adding 2× NuPAGE LDS sample buffer and then incubated for 5 min at 95 °C. Samples were resolved by SDS-PAGE and analyzed by standard western blotting techniques. Antibodies against CHK1 (2G1D5; Cell Signaling Technology), phospho-CHK1 (S345), β-actin (Sigma-Aldrich) and phospho-RPA32 (S4/8) were used. Signals were developed using an Enhanced Chemiluminiscence Kit (Amersham-Pharmacia Biotech, Buckinghamshire, UK), according to the manufacturer’s instructions.
Chk1 2g1d5
Manufactured by Cell Signaling Technology
Sourced in United States
CHK1 (2G1D5) is a laboratory reagent produced by Cell Signaling Technology. It is an antibody that recognizes the Checkpoint Kinase 1 (CHK1) protein, which plays a crucial role in DNA damage response and cell cycle regulation.
Automatically generated - may contain errors
Lab products found in correlation
Fancd2, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminiscence kit, by Cytiva (1 mentions)
β actin, by Merck Group (1 mentions)
Ab213, by Abcam (1 mentions)
Ab2187, by Abcam (1 mentions)
Brca1, by Santa Cruz Biotechnology (1 mentions)
Fancd2, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Cyclin a, by GeneTex (1 mentions)
Vinculin, by Merck Group (1 mentions)
Palb2, by Fortis Life Sciences (1 mentions)
Alexa fluor, by Thermo Fisher Scientific (1 mentions)
Ph2ax, by Merck Group (1 mentions)
Ubiquityl pcna lys164, by Cell Signaling Technology (1 mentions)
Fanci, by Santa Cruz Biotechnology (1 mentions)
Phospho atr ser428, by Cell Signaling Technology (1 mentions)
Pcna pc10, by Cell Signaling Technology (1 mentions)
Beta actin, by GeneTex (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
4 protocols using chk1 2g1d5
1
Western Blot Analysis of DNA Damage Signaling
2
Western Blotting and Immunofluorescence Analysis
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The antibodies used for Western blotting were as follows: FANCM (sc-98710), FANCD2 (sc-20022), BRCA1 (sc-642), β-actin (sc-47778; Santa Cruz, CA), RPA (NA18; Calbiochem, CA), phosphor-RPA(S4/S8) (A300-245A; Bethyl Lab), CHK1 (2G1D5; Cell Signaling, MA) and phosphor-CHK1(S317) (AF2054; R&D System, MN). FANCD2 foci were detected by immunofluorescence staining using an anti-FANCD2 antibody (AB2187; Abcam). γ-H2AX foci were visualized by immunofluorescence staining using anti-γ-H2AX mouse monoclonal antibody (JBW301; Upstate Biotechnology, NY). RAD51 foci were detected by immunofluorescence staining using mouse monoclonal anti-RAD51 (14B4) (ab213; Abcam, MA).
3
Western Blot Analysis of DNA Repair Proteins
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Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used. Secondary antibodies were conjugated with horse radish peroxidase (mouse or rabbit, Cell Signaling Technology) or appropriate Alexa Fluor (Molecular Probes) were used.
4
Quantitative Protein Analysis by SDS-PAGE and Western Blot
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Total protein was extracted from exponentially growing cells at passage 8–10 and 40 g/mL were resolved by SDS-PAGE using a 4–15% gradient gel (Bio-Rad Laboratories). After transfer and blocking overnight at 4 °C in Odyssey Blocking Buffer (Li-Cor, Lincoln, NE, USA) proteins were detected by primary antibodies against BRCA1 [2A-9] (1:500, kindly provided by Stephen Smith, Leibnitz Institute, Jena, Germany), ATR [N-19] (Santa Cruz, St. Cruz, CA, USA, 1:1000), CHK1 [2G1D5] (Cell Signaling, Danvers, MA, USA, 1:750), RAD51 [14B4] (1:2.000, GeneTex, Irvine, CA, USA), MRE11A [12D7] (Abcam, Cambridge, UK, 1:500), pCHK1 [Ser296] (Cell Signaling, 1:1000), -actin [AC-74] (1:50.000, Sigma, St. Louis, MO, USA). Primary antibodies were detected with IRDYE 680 conjugated anti-mouse IgG, IRDYE 800 conjugated anti-rabbit IgG (Li-Cor, 1:7500), IRDYE 680 conjugated anti-rabbit IgG (Licor, 1:7.500 or 15.000) or IRDYE 800 conjugated anti-mouse IgG (Li-Cor 1:7.500 or 15.000). Quantitative and qualitative analysis was done by using Li-Cor Odyssey (Li-Cor, Lincoln, NE, USA).
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