Anti h3k4me2

Total protein was isolated from a pool of 10 cultured cochleae in each group using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. A BCA protein kit (Beyotime, Jiangsu, China) was used to measure the protein concentrations. After separation by 12% SDS-PAGE, the proteins were blotted onto PVDF membranes (Immobilon-140 P; Millipore, Bedford, MA). The membranes were blocked with 5% nonfat dried milk in TBST (50 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature and hybridized overnight at 4 °C with the primary antibodies. Antibodies included anti-H3K4me2 (1:1000 dilution; Abcam), anti-cleaved caspase-3 (1:500 dilution; Cell Signaling Technology, Inc.), and anti-GAPDH (1:5000 dilution; Cell Signaling Technology, Inc.). The membranes were subsequently washed three times in Tris-buffered saline with Tween-20 (TBST) for 10 min each and then incubated with HRP-conjugated secondary antibody diluted 1:5000 (Supersignal West, Pierce, Rockford, IL) for 1 h at room temperature. Immunoreactive bands were visualized using the ECL-Kit according to the manufacturer’s instructions (Pierce).

The co-IP assay was performed as previously described. Briefly, MDA-MB-231 cells were seeded at the density of 1 × 10⁶ cells in a six-well plate. Cells were treated with the indicated concentrations of 1 for 24 h under 37 °C 5% CO₂. Cells were lysed and collected the protein samples. The concentration of protein samples was calculated using the Pierce BCA protein assay kit. 30 μg of each protein sample were incubated overnight with 10 μL pre-incubated anti-KDM5A magnetic beads according to the manufacturer’s protocol. The complex was washed 5 times to elute non-specific and non-cross-linked antibodies. Then, the precipitated proteins were subjected to SDS-PAGE and analyzed by Western blotting with anti-H3, anti-H3K4me2, and anti-H3K4me3 (1:1000, Abcam).