Anti ezh2 antibody

ISH and IHC were performed and subsequent results were analyzed as previously described [29 (link)]. In brief, the triple digoxigenin-labeled antisense locked nucleic acid (LNA)-modified probes for HOTAIR and miR-193a were synthesized by Boster Biotech (Wuhan, China). ISH was conducted according to the manufacturer’s instruction of the HOTAIR and microRNA ISH Optimization Kits (Boster, Wuhan, China). IHC was performed using anti-EZH2 antibody (1:250, Abcam, Cambrige, MA, USA) according to the manufacturer’s instructions. The original magnification: ×200.The specific evaluation of gene expression via ISH or IHC was calculated as previously described [30 (link)]. In brief, sections with no labeling or less than 5% labeled cells were scored as 0, 5%–30% of cells as 1, 31%–70% of cells as 2, and ≥71% as 3. The staining intensity was scored similarly using 0 for negative staining, 1 for weakly positive, 2 for moderately positive, and 3 for strongly positive. The scores of percentage of positive tumor cells and staining grade were calculated to generate the immune-reactive score for each specimen. A combined score of 0–1 indicates negative expression (−), 2–3 indicates weak expression (+), 4–5 indicated moderate expression (++), and 6 indicates strong expression (+++). Each sample was examined separately and scored by two pathologists [30 (link)].

According to the instructions of the Magna RIP RNA-binding protein immunoprecipitation kit purchased from Millipore, the RIP assay was conducted to assess the interaction between EZH2 and LATS2-AS1-001. Total RNA was precipitated with anti-EZH2 antibody (Abcam, USA). Then, qRT-PCR was performed to measure the enrichment of immunoprecipitated RNAs.

The ChIP assay was performed using the EZ ChIP Chromatin Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Briefly, 1 × 10⁷ cells were cross-linked with 1% formaldehyde at room temperature for 10 min, followed by fragmentation of the chromatins to the size of 200–500 bp fragments using an ultrasonication. The DNA–protein complexes were immunoprecipitated using anti-EZH2 antibody, anti-H3K27me3 antibody, or anti-IgG antibody (1:100; Abcam, San Francisco, USA). The ChIP-derived DNA was subsequently amplified by qPCR method, and the percentage of input DNA was calculated. The Primers for the RASA1 promoter are listed in Additional file 2: Table S2.

RIP assay was used to evaluate binding between STXBP5-AS1 and EZH2. The assay was performed with the EZ-Magna RIP Kit (Millipore, MA, USA) following the manufacturer’s manual. Anti-EZH2 antibody and control IgG were obtained from Abcam. The immunoprecipitated RNA was recovered and further analyzed by qRT-PCR as previously described.

RIP assay was conducted using EZ-Magna RIP kit (Millipore, MO, USA) in accordance with the provider’s guide. The anti-EZH2 antibody (Abcam, Cambridge, UK) and control IgG was used. The RNA species in EZH2 immunoprecipitate complex was measured by qPCR.