SH-SY5Y cells were treated with 0.2 μM dPPA for 72 h, and then, ChIP analysis was performed using ChIP assay kits (Beyotime) according to the manufacturer's recommendations with a CEBPβ antibody (Santa Cruz Biotechnology, sc-9314) and control IgG (Abcam). The input control DNA or immunoprecipitated DNA was then subjected to PCR amplification using primers specific to the furin promoter (forward primer 5′-ACCAGAGCCCAGCGTTCAGCAG-3′ and reverse primer 5′-CATGGCCAGCCAGTGCTGCCA-3′). The PCR products were separated by 2% agarose gel electrophoresis and visualized with ethidium bromide staining.
C ebpβ antibody
Manufactured by Santa Cruz Biotechnology
Sourced in Germany
The C/EBPβ antibody is a protein-specific antibody that recognizes the C/EBPβ (CCAAT/enhancer-binding protein beta) transcription factor. C/EBPβ is a member of the C/EBP family of basic leucine zipper (bZIP) transcription factors and plays a key role in the regulation of genes involved in cell growth, differentiation, and inflammatory responses.
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Lab products found in correlation
Control igg, by Abcam (1 mentions)
Chip assay kit, by Beyotime (1 mentions)
Chip kit, by Merck Group (1 mentions)
Protein a agarose salmon sperm dna, by Merck Group (1 mentions)
Caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Antibody against gapdh, by Santa Cruz Biotechnology (1 mentions)
Zbtb7a antibody, by Santa Cruz Biotechnology (1 mentions)
Il 24, by Proteintech (1 mentions)
4 protocols using c ebpβ antibody
1
Regulation of Furin Expression by CEBPβ
2
ChIP-qPCR Protocol for C/EBPβ Binding
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The ChIP assay was performed using ChIP kit (Merck, Germany) with C/EBPβ antibody (Santa Cruz Biotechnology, USA) (30 (link)). Cells in a 10-cm culture plate were crosslinked with 1% formaldehyde for 10 minutes. Crosslinking was neutralized with 0.2M glycine. Cells were collected and suspended in lysis buffer. Genomic DNA was released by using SDS Lysis Buffer, and sonicated to 200-1000 bps. Protein–DNA complexes were precipitated with C/EBPβ antibody (Cell Signaling Technology, USA). After purification, the precipitated DNA and input were de-crosslinked at 65°C and then purified. The capacity of C/EBPβ binding to CLTA and CLTC promoter were quantified by qPCR. Primer sequences used in ChIP-qPCR were as follows: CLTA sense 5’-ATGGCCCAGATGGAGAAAGC-3’ and antisense 5’-GGGAGGTGTTGGATGTGAGG-3’; CLTC sense 5’-ATGGCCCAGATGGAGAAAGC-3’ and antisense 5’-TGTTTCGACTGAGCCCCT-3’.
3
ChIP Assay for C/EBPβ Binding to KLF4 Promoter
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The binding of C/EBPβ to the KLF4 promoter after treatment with or without EPHA7 was measured with ChIP assay referring to previous protocol [39 (link)]. The lysed KGN cells were sonicated and pre-cleaned with Protein A Agarose/Salmon Sperm DNA (Millipore). Then the sheared chromatin DNA was immunoprecipitated with C/EBPβ antibody (Santa Cruz Biotechnology) or negative control IgG, followed by washing with Magna ChIP Protein A Agarose Magnetic Beads (Millipore). After reverse cross-linking, RNA contamination and protein digestion, sheared DNA was extracted using a DNA extraction kit for next quantitative analysis. The primer sequences of KLF4 used for qRT-PCR were 5′-CGAACGCCGGAATCCAAAGT-3′ (forward) and 5′-TCCCCTTGGTTTGTGATCAGT-3′ (reverse), which amplified a region between −879 bp and − 865 bp spanning the putative C/EBPβ binding site. The ratio of DNA precipitated by C/EBPβ antibody over input control indicated the amount of bound transcription factor.
4
Osteosarcoma Cell Lines and Antibody Analysis
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The human osteosarcoma cancer cell lines U2OS and MG63 were obtained from the American Type Culture Collection. U2OS cells were cultured in DMEM with 10% fetal bovine serum (FBS; ExCell Bio, Lot: FSP500). MG63 cells were cultured in EMEM medium with 10% fetal bovine serum (FBS; ExCell Bio, Lot: FSP500). The medium was renewed every day, and cells were passaged before reaching confluence. The following antibodies were used in this study: antibody against GAPDH (Santa Cruz Biotechnology, Dallas, TX; SC-25778); caspase 3 antibody (Cell Signaling Tech, 9662); PARP (Santa Cruz Biotechnology, SC-8007); ZBTB7A antibody (Santa Cruz Biotechnology, SC-33683); C/EBPβ antibody (Santa Cruz Biotechnology, SC-150); C/EBPα antibody (Santa Cruz Biotechnology, SC-9351); IL24 (Proteintech, 26,772–1-AP); and cisplatin (Sigma, P4394).
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