Human anti ezh2 antibody

RNA immunoprecipitation was performed using thermo fisher RIP kit (Thermo, USA) based on the manufacturer’s protocol. Cells at 80–90% confluency were scraped off and lysed in complete RIP lysis buffer. Then, 100 μl of whole cell extract was incubated with RIP buffer containing magnetic beads conjugated with human anti-EZH2 antibody (Cell Signaling, USA), anti-SUZ12 antibody (Cell Signaling, USA), anti-EED antibody (Cell Signaling, USA) negative control normal mouse IgG (Millipore).

RIP experiments were performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Briefly, FRO and 8505c cells were lysed in RIP lysis buffer. First, 5 µg of human anti-EZH2 antibody (#4905, Cell signaling, Danvers, MA, USA) and normal rabbit IgG (Millipore) used as negative control, were incubated with magnetic beads for 30 min. Then, 100 μL of whole lysates were incubated overnight on a rocking platform at 4 °C. The next day, samples were incubated with Proteinase K buffer at 65 °C for 30 min and then immunoprecipitated RNA was purified. Purified RNA was reverse transcribed into cDNA by using random primer with the QuantiTect Reverse Transcription Kit (Qiagen), and the presence of PAR5 transcripts was detected by qRT-PCR. Enrichment of PAR5 transcripts in the EZH2 immunoprecipitated lysate was calculated relative to the levels of PAR5 transcript in the rabbit IgG control sample, set equal to 1 [51 (link),52 (link)].