Anti cbx3

RNA-ChIP was performed using the EZ-Magna RIP kit (Millipore) per the manufacturer's instructions as previously described. In brief, 10⁷ cells were lysed with RIP lysis buffer with one freeze-thaw cycle. Cell extracts were coimmunoprecipitated with anti-TCEA1, anti-RBMX, anti-MCM2 or anti-CBX3 (Abcam), and the retrieved RNA was subjected to real-time qPCR analysis. Normal rabbit IgG was used as a negative control.

IHC was performed on tissue microarrays of GC (Outdo Biotech Co., Shanghai) following a previous study.14 (link) We used anti-CBX3 (Abcam) as the primary antibody. Immunoreactivity was determined by a semiquantitative method according to the staining intensity of cancer cells and the positively stained proportion. The scoring criteria for proportion of positively-staining tumor cells were as follows: 0, none; 1, 10%; 2, 11–50%; 3, 51–80%; and 4, >80%. The scoring criteria for the staining intensity: 0, no staining; 1, weak; 2, moderate; and 3, strong. We obtained the semiquantitative immune score by multiplying the positive cell score by the staining intensity score ranging from 0 to 12. This study was approved by the Ethical Review Board of the First Affiliated Hospital of Xiamen University.