Abe1867

The ChIP assay was carried out as per the specifications of the EZ ChIP Kit (Merck Millipore, Darmstadt, Germany). PANC-1 cells transfected for 48 h underwent 10-min cross-linking with 1% formaldehyde, which was terminated with glycine. The cell precipitate was lysed with SDS lysis solution (SDS Lysis Buffer + Proteinase Inhibitor Cocktail II) at a ratio of 100 μL per 10^{6 (link)} cells, and then the cell lysates were placed on a sonicator to cut DNA into fragments of 200–1000 bp in length. ChIP assay was performed with IgG antibodies (Merck Millipore), KMT2D polyclonal antibodies (ABE1867, Merck Millipore), and H3K4Me1 monoclonal antibodies (ab176877, Abcam). The purified DNA products were subjected to RT-qPCR to confirm whether the DNA precipitated by the antibodies contained the DNA sequence and the relative content of the target gene.

The cells were lysed with pre-cooled RIPA lysis solution and centrifuged at 4°C and 14,000 g for 15 min, with the supernatant transferred to new centrifuge tubes. The lysates (1 mL) were added with anti-ASH2L (A300-112A, Thermo Fisher Scientific, Rockford, IL, USA), anti-KMT2D (ABE1867, Merck Millipore), or IgG antibodies (ab172730, 1:100, Abcam; as the NC), and the antigen–antibody mixtures were slowly shaken at 4°C overnight or at room temperature for 2 h on a shaker. Then, 100 μL Protein A/G agarose beads (prepared with PBS solution to a concentration of 50%) were added to capture the antigen–antibody complexes. The antigen–antibody mixtures were slowly shaken at 4°C overnight on a shaker or incubated at room temperature for 1 h. Subsequent to 5-s instantaneous centrifugation at 14,000 rpm, the supernatant was removed, and the agarose bead-antigen-antibody complexes were harvested. The complexes were washed thrice with 800 μL pre-cooled RIPA buffer, suspended with 60 μL of 2 × loading buffer, and gently tapped for mixing. The loading samples were boiled for 5 min and centrifuged at 14,000 g, and the remaining agarose beads were attained. The supernatant was denatured through 5-min boiling and then subjected to electrophoresis, with the protein expression assessed by reference to the procedure of western blot described above.