Ab109489

Pancreatic cancer cells of 70–80% fusion were added with 1% formaldehyde, and fixed at room temperature for 10 min to fix and cross-link the DNA and protein in cells. After cross-linking, cells were randomly broken by ultrasonic treatment for 10 s each time, with time interval of 10 s, in a total of 15 cycles, so as to break the cells into fragments of appropriate size. After centrifugation at 4°C (13,000 rpm), the supernatant was collected and divided into three tubes, with the addition of RNA polymerase II (ab109489, 1:100, Abcam, United Kingdom) as the positive control antibody, the IgG (ab109489, 1:100, Abcam, United Kingdom) as the negative control antibody of normal mice and the mouse anti-H3K27me3 (1:100, ab4729, Abcam, United Kingdom) as the target protein specific antibody, followed by overnight incubation at 4°C. The endogenous DNA protein complex was precipitated by Protein Agarose/Sepharose, the supernatant was removed after transient centrifugation, and the non-specific complex was washed, followed by overnight cross-linking at 65°C. Then, the DNA fragments were extracted, purified and recovered by using phenol/chloroform. Finally, the binding of CDKN1C to H3K27me3 was detected by CDKN1C promoter specific primers.

Clinical breast cancer tissues were analyzed by immunohistrochemistry (IHC) to analyze IGHG1 expression as previously described [11 (link)]. Deparaffinized sections were subjected to antigen retrieval by microwave heating in 10 mM citrate buffer. Fixation was performed using paraformaldehyde. Cells were permeabilized, blocked, and labeled with anti-IGHG1 (1:1000, ab109489, Abcam, Shanghai, China). Immunostained tissues were analyzed using the HRP detection system.

When confluence reached 70‐80%, cells were fixed with 1% formaldehyde at room temperature for 10 minutes for crosslinking cell DNA and protein. Then the cells sonicated for 10 seconds, 15 cycles. After centrifugation at 13 000 rpm, the supernatant was divided into two parts and respectively incubated with anti‐rabbit IgG (ab109489, 1:100, Abcam) and target protein specific anti‐mouse ETS1 (Cell Signaling: #14069). Protein Agarose/Sepharose was added to precipitate endogenous DNA‐protein complex, followed by centrifugation and decrosslinking. DNA fragments were purified with Phenol/Chloroform. ITPR1 enhancer specific primer: Forward: GCGTCCAGTGACCAGGG, Reverse: TTAAAGCGGCTCCGGGTG was used to evaluate the enrichment of ETS1 in ITPR1 enhancer. Same procedure was performed to detect enrichment of SLC26A4‐AS1 in ETS1 enhancer.