MC3T3-E1 cells were expanded until confluent and treated with either 2.5 μM NE or vehicle control for 48 hours. DNA was subsequently extracted. All manufacturer instructions were subsequently followed for chromatin immunoprecipitation (CHiP) assay (Qiagen, cat. #334471). Antibodies were used for ATF4 (Cell Signaling, cat. #D4B8) and CREB1 (Cell Signaling, cat. #9197T). Primers were used for ATF4 (ThermoFisher, cat. #Mm00515325) and CREB1 (ThermoFisher cat. #Mm00501607). Data are presented as differences in promoter region amplification relative to IgG control and were statistically compared using two-way ANOVA with Sidak's multiple-comparisons test.
Chip assay
Manufactured by Qiagen
The ChIP assay is a laboratory technique used to investigate the interactions between proteins and DNA in a cellular context. It allows researchers to identify the specific DNA sequences that are bound by a particular protein of interest.
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3 protocols using chip assay
1
ATF4 and CREB1 Binding in MC3T3-E1 Cells
2
Investigating E2F1 Regulation of CDCA5 via ChIP
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293T cells were seeded in a 24-well plate and transfected with E2F1-Flag or vector plasmid. After 48 h, cells were collected for ChIP. ChIP assay (Qiagen, German) was conducted according to the manufacturer's instructions. Flag antibody (Sigma, USA) or IgG antibody (CST, USA) was used for immunoprecipitation. DNA was quantified using PCR. Primers are listed below: ChIP-CDCA5-1: 5'-CACCACTACGCTCTCACCAG-3', 5'-CAGGAATCTGAGATCGGGGC-3'; ChIP-CDCA5-2:5'-GGAAAGGGGCATGAGTTGGTA-3', 5'-GGCCACCAGAACCTGACTTC-3'; ChIP-CDCA5-3: 5'-GACCCGATGTTCGAAGCAGA-3', 5'-CAACTCATGCCCCTTTCCCA-3'; ChIP-CDCA5-4: 5'-CACCTTGGCGTGATTGGCTA-3', 5'-TACCAACTCATGCCCCTTTCC-3'.
3
FOXO1 Regulation of MALAT1 Promoter
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FOXO1 Binding Sites on MALAT1 gene promoter have been identified on chr11: 65255303-65255313 and binding sequence: TCCTGTTTATG by ChIP assay (QIAGEN). The chromatin-protein complex from the cells was isolated by immunoprecipitation of the chromatin with FOXO1 antibody (ab39670, Abcam, Cambridge, MA, USA) using Magna-ChIP assay kit (Cat# 17-10085, Sigma-Aldrich) following the manufacture’s instruction and then qPCR with MALAT1 promoter primers. The primer covering the FOXO1-binding region of the promoter of MALAT1 was designed and synthesized by QIAGEN. Fold enrichment was calculated as the amount precipitated by anti-FOXO1 IgG relative to the amount precipitated by normal IgG (mock samples). The data were also calculated as % input to verify that results were consistent.
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