H3k27me3 antibody

Splenocytes were obtained from BALB/c mice according to the conventional method (57 (link)) and were suspended in a 24-well plate (1 × 10⁷ cells/well). All mouse splenocytes were divided into three groups (normal control, viral control, and umifenovir treatment group). The viral control and umifenovir treatment group were infected with CVB4 (1.4 × 10⁶ TCID₅₀), and the umifenovir treatment group was treated with umifenovir (12 μM) additionally. Then the three groups of splenocytes were collected after 48 h in culture for the subsequent chromatin immunoprecipitation (ChIP) experiment.
ChIP assays were performed using a ChIP kit (Abcam, MA) according to the manufacturer’s protocol. Three groups of splenocytes (1 × 10⁶ cells/group) were collected and fixed with 1% formaldehyde for 10 min at room temperature. Then SDS nuclear lysis, ultrasonic DNA shearing, protein and DNA immunoprecipitation, cross-linked DNA reversal, and DNA purification were conducted step by step. Anti-H3K9me3, -H3K9ac, and -H3K27me3 antibodies (Abcam) were used for coimmunoprecipitation, and mouse IgG (Abcam) was used for the negative control. The purified coprecipitated DNA fragments were used to detect fragments from IL-10 promoter (F, 5′-GAGACGTGTAACCTGTAGCT-3′, and R, 5′-GTAGGAGAAGTCCCTACTGA-3′) by quantitative real-time PCR (RT-qPCR).

The EpiQuik Total Histone Extraction Kit (Epigentek) was used to extract histones from honeybee ovary tissue. Qubit fluorometer and protein assay kits (Invitrogen) were used to estimate protein concentration. Ten micrograms of protein were separated on a 12% SDS PAGE gel at 200 V for 1 h in Tris-Glycine-SDS electrophoresis buffer and run alongside a molecular weight marker (Novex prestained ladder [Invitrogen]). Proteins were transferred to nitrocellulose membrane in Towbin’s buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3, 20% methanol), the membrane was blocked in 2% bovine serum albumin in TBS-T before incubation with H3K27me3 antibody (Abcam 6002) (1:200) at 4 °C overnight. The membrane was washed and incubated with HRP-conjugate anti-mouse (Jackson Immunochemicals) (1:1,000) at room temperature for 1 h. The chemiluminescent reaction step was performed using the Pierce ECL Western Blotting substrate (Thermofisher). The blot was imaged using the Fuji LAS-3000 ECL imaging system. After detection of H3K27me3, the membrane was stripped for 10 min in stripping buffer (200 mM Glycine, 0.01% SDS, 0.1% Tween) and washed before incubation with the anti-Histone H3 Polyclonal Rabbit (abcam 1791) (1:1,000) and a second chemiluminescent reaction step.

PC3 and DU145 cells which were transfected with si-NC or si-HOTAIR2 for 72 h and processed with ChIP assay kit according to the manufacturer’s instructions (Beyotime, China). Antibodies used includes EZH2 antibody (1:50, Abcam, Cambridge, MA, USA), H3K27me3 antibody (1:50, Abcam, Cambridge, MA, USA) and IgG (1:50, Millipore, Billerica, MA, USA). Gene Expression Omnibus (GEO) datasets (DU145 H3K27me3: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1138596; PC3 H3K27me3: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1383872) were retrieved and analyzed for the H3K27me3 peak loci at miR-193a promoter region. ChIP qPCR Primers were specifically designed according to the H3K27me3 loci in PCa cells. The precipitated DNA quantitated by qRT-PCR and normalized by respective 1% ChIP input. ChIP primer sequences used are as follows: sense, 5′-AAAGGCATGATCTGGGTTGGT-3′; antisense, 5′-AGGTCGAGATTTGGAGCCATTTA-3′.