Protocol detail

ChIP-qPCR Analysis of PD-L1 Promoter

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Soluble chromatin was prepared from 3×10⁶ cells and precipitated with anti-MYC, anti-NF-κB p65 (Santa Cruz Biotechnology), anti-MUC1-C or a control nonimmune IgG. Power SYBR Green PCR Master Mix (Applied Biosystems) and ABI Prism Sequence Detector (Applied Biosystems) were used for amplification of ChIP qPCRs. Primers used for qPCR of the PD-L1 promoter and GAPDH control region are listed in Supplementary Table S2. Relative fold enrichment was calculated as described (26 (link)).

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Maeda T., Hiraki M., Jin C., Rajabi H., Tagde A., Alam M., Bouillez A., Hu X., Suzuki Y., Miyo M., Hata T., Hinohara K, & Kufe D. (2017). MUC1-C INDUCES PD-L1 AND IMMUNE EVASION IN TRIPLE-NEGATIVE BREAST CANCER. Cancer research, 78(1), 205-215.

Publication 2017

Power SYBR Green PCR Master Mix ABI Prism Sequence Detector

Cells Chip Chromatin Gapdh Muc1 Nf κb p65 Pd l1 Primers Prism Sybr green

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Variable analysis

independent variables

Anti-MYC
Anti-NF-κB p65 (Santa Cruz Biotechnology)
Anti-MUC1-C
Control nonimmune IgG

dependent variables

Enrichment of PD-L1 promoter

control variables

GAPDH control region
Power SYBR Green PCR Master Mix (Applied Biosystems)
ABI Prism Sequence Detector (Applied Biosystems)

controls

Positive control: Not specified
Negative control: Control nonimmune IgG

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