Human anti ezh2 antibody

RNA immunoprecipitation was performed using the EZ-Magna RIP kit (Millipore, China) following the manufacturer's guidance. 2 × 10⁷ hMSCs at 80–90% confluency were lysed in 2 ml PBS, 2 ml nuclear isolation buffer (1.28 M sucrose; 40 mM Tris-HCl pH 7.5; 20 mM MgCl2; 4% Triton X-100) and 6 ml ddH₂O. Nuclei was resuspended in RIP buffer, sonicated and incubated with magnetic beads conjugated with human anti-EZH2 antibody (Abcam, China), negative control normal mouse IgG (Abcam, China) overnight followed by stringent washing of protein A/G bead pellets. Samples were incubated with Proteinase K with shaking to digest the protein and then immunoprecipitated RNA was isolated. Furthermore, purified RNA was subjected to qRT-PCR analysis to demonstrate the presence of HoxA-AS3.

RIP experiments were performed using the EZ‐Magna RIP kit (Millipore, China) following the manufacturer's guidance. Human anti‐EZH2 antibody (Abcam, Shanghai, China), SUZ12 (Santa Cruz, Shanghai, China), LSD1 (Santa Cruz), or control IgG was used. The immunoprecipitated RNA was isolated and was further subjected to qRT‐PCR analysis.

The treated cells were collected, and their nuclear proteins were extracted and then resuspended in RIP buffer. Then divide the resuspended RIP buffer into the input group, IgG group, and EZH2 group. Then collect the supernatant after centrifugation, add IgG (Abcam) or human anti-EZH2 antibody (Abcam), and then incubate at 4 °C for 2 h. Subsequently, protein A beads were added and incubated at 4 °C for 1 h. After centrifugation, the cells were washed three times with RIP buffer and then once with PBS, and the beads were resuspended in Trizol. Finally, quantitative detection was performed by qRT-PCR.