5–10×106 RD cells were plated as spheres for 24–48 hours. Spheres were then cross-linked with 1% formaldehyde for 10–15 min, quenched with 0.125M glycine for 10min, and washed with PBS. Cells were resuspended in lysis buffer (50mM Tris pH7.5, 150mM NaCl, 5mM EDTA, 1% Triton-X, 0.1% SDS, 0.5% sodium deoxycholate) and sonicated (Misonix XL-2000) for 14 cycles (12 sec on, 2 min off). Cell debris was pelleted, and chromatin was precleared with Protein G agarose beads (Millipore) for 2 hours at 4°C. RBPJ antibody (Cell Signaling #5313) was added at 1:50 and rotated overnight at 4 °C. Protein G beads were added the next day for 3 hours with rotation at 4 °C. Beads were washed according to the Abcam protocol and DNA was eluted with elution buffer (Santa Cruz) at 67°C for 2 hours with rotation. Crosslinks were reversed overnight followed by a proteinase K digestion for 1hr. DNA was purified using the Qiagen PCR Purification kit. ChIP enrichment was evaluated using semi-quantitative PCR followed by quantitation using ImageJ (NIH) similar to previous published work (57 (link)). ChIP primers are listed in Supplementary Table 1 .
Rbpj antibody
Manufactured by Cell Signaling Technology
The RBPJ antibody is a research-use only product manufactured by Cell Signaling Technology. It is designed to detect RBPJ (Recombination Signal Binding Protein For Immunoglobulin Kappa J Region) protein levels in various sample types. RBPJ is a transcription factor that plays a key role in the Notch signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Normal rabbit igg, by Cell Signaling Technology (2 mentions)
Elution buffer, by Santa Cruz Biotechnology (1 mentions)
Xl 2000, by Bioventus (1 mentions)
Protein g agarose beads, by Merck Group (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Notch1 antibody, by Abcam (1 mentions)
Recombinant mouse dll4, by BioLegend (1 mentions)
3 protocols using rbpj antibody
1
RBPJ Chromatin Immunoprecipitation Protocol
2
Chromatin Immunoprecipitation of Notch Signaling Components
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For Figure 4C , cell culture plates were pre-coated with PBS or 5 μg/ml recombinant mouse DLL4 (BioLegend, #776706) overnight at 4°C before seeding EL4 cells. For Figure 4B,F–I,K and L , soluble DLL4 (5 μg/ml) was used. EL4 cells were incubated with soluble DLL4 for 24 hr before crosslinked with 1% formaldehyde for 10 min at room temperature. Reaction was quenched with 125 mM glycine. ChIP was performed as previously described (Zhao et al., 2018 (link)) with NOTCH1 NICD antibody (Abcam, ab27526), RBPJ antibody (Cell Signaling Technology, #5313), or normal rabbit IgG (Cell Signaling Technology, #2729). The sequences of all ChIP primers are listed in Appendix 1.
3
Investigating NOTCH1 and RBPJ Binding in EL4 Cells
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EL4 cells were treated with PBS or 5 µg/ml recombinant mouse DLL4 (BioLegend, #776706) for 24 hours before crosslinked with 1% formaldehyde for 10 minutes at room temperature.
Reaction was quenched with 125 mM glycine. ChIP was performed as previously described (Zhao et al., 2018) with NOTCH1 antibody (Abcam, ab27526), RBPJ antibody (Cell Signaling Technology, #5313), or normal rabbit IgG (Cell Signaling Technology, #2729). The sequences of all ChIP primers are listed in Supplemental Table 2.
Reaction was quenched with 125 mM glycine. ChIP was performed as previously described (Zhao et al., 2018) with NOTCH1 antibody (Abcam, ab27526), RBPJ antibody (Cell Signaling Technology, #5313), or normal rabbit IgG (Cell Signaling Technology, #2729). The sequences of all ChIP primers are listed in Supplemental Table 2.
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