Anti kdm2b

Tumor cells (2 × 10⁶) were prepared for chromatin immunoprecipitation (ChIP) assay with the ChIP assay kit (Millipore Technology, Billerica, MA) according to the manufacturer’s protocol. The resulting precipitated DNA samples were analyzed using quantitative real-time PCR to amplify a 408-bp region of the MOB1 promoter with the primers 5′-CGC TTA TAG AGG TCC CTG CAT AAC-3′ (forward) and 5′-TCA GGA TTC GGA GCT GGC TAG-3′ (reverse); a 80-bp region of the YAP promoter with the primers 5′-TCG CCG CTT CTC CAC CT-3′(forward) and 5′- GAC GCG CAC CCC CTG AC-3′ (reverse); a 101-bp region of the TAZ promoter with the primers 5′- AGA AAC CTC AGA GTG ACT AAA AT − 3′(forward) and 5′- TAA GAG TCA GAT GAG CAG AGA TG − 3′ (reverse). The antibodies used in the ChIP assay were anti-KDM2B (#09–864, Millipore), anti-H3K27me3 (#9733, Cell Signaling Technology), anti-H3K36me2 (#2901, Cell Signaling Technology), anti-H3K4me3 (#9751, Cell Signaling Technology). The experiments were performed in triplicate and repeated twice.

Neuro-2a were grown in MEM containing 10% FBS for 24 h and incubated with 1% formaldehyde for 10 min at 37°C. Cells were collected, lysed, and sonicated during 10 min at medium intensity in the ultrasonic processor GEX-600 (Sonics & Materials) and treated for ChIP as recommended by the manufacturer (Upstate). We used a rabbit anti-KDM2B (Millipore) and the unrelated rabbit anti-βIII tubulin (Sigma) antibodies. For PCR analysis, we used ChIPCK forward primer (5´-AGTTTTTGGCTTCCAGCAGA-3´) and ChIPCK reverse primer (5´-ACATTAGTCATGGTCACGCG-3´) [10 ]. PCR was performed using 5 μl of template DNA, 1.5 mM MgCl₂, and 20 pmol of each primer for 35 cycles at 94°C for 30 seg, 60°C for 30 seg, and 72°C for 30 seg.