Anti ezh2 antibody

Spleen samples were homogenized in RIPA buffer (Santa Cruz Biotechnology, CA) with proteinase and phosphatase inhibitors (Roche, Indianapolis, IN). Proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore, Billerica, MA), incubated with anti-Ezh2 antibody (Cell Signaling Technology, Boston, MA), and subsequently with secondary anti-rabbit IgG antibodies labeled with HRP, both in blocking buffer (1% dry milk). Proteins were visualized with enhanced chemiluminescence substrate (Fisher Scientific, Hampton, NH).

Whole proteins were extracted and immunoprecipitated using an anti-EZH2 antibody (#5246, Cell Signaling Technology). The precipitated samples were then subjected to western blot analysis. The membrane was sequentially probed with anti-phospho-threonine (#9381, Cell Signaling Technology) and anti-EZH2 (Cell Signaling Technology) antibodies.

Paraffin-embedded tissue blocks were sectioned, deparaffinized in xylene and rehydrated for immunohistochemical staining [83 (link)]. Antigen retrieval was performed using sodium citrate. The sections were then incubated in H₂O₂ (3%) for 10 min, blocked in 1% bovine serum albumin for 60 min and incubated with an anti-EZH2 antibody (Cell Signaling Technology, 1:400) at 4°C overnight. After incubation with a secondary antibody for 60 min, the specimens were incubated with H₂O₂-diaminobenzidine until the desired staining intensity was observed. The sections were counterstained with hematoxylin, dehydrated and mounted. Immunohistochemical staining was evaluated in accordance with the immunoreactive score (IRS), in which IRS = staining intensity (SI) X percentage of positive cells (PP). SI was scored as follows: negative SI = 0; weak SI = 1; moderate SI = 2; and strong SI = 3. Similarly, PP was scored as follows: negative PP = 0; 10% PP = 1; 11–50% PP = 2; 51–80% PP = 3; and >80% PP = 4. All specimens used for immunohistochemical staining were evaluated and scored by at least two independent pathologists.

RNA EMSA was performed using LightShift Chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific, Shanghai, China). Briefly, nuclear extracts were isolated from PT-U cells with NEPER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer’s instructions. Biotin-labeled RNA probes and unlabeled complementary RNA fragments were obtained by in vitro transcription assays with Biotin RNA Labeling Mix (Roche, Basel, Switzerland). Protein-lncRNA binding reactions were performed in proteins of PT-U cells along with Biotin-labeled RNA probes or unlabeled complementary RNA fragments. After incubation in 1 × EMSA binding buffer, the components were separated with native PAGE and then transferred on to positively charged nylon membrane (Roche, Mannheim, Germany). After UV cross-linking, biotin signals were detected with HRP–conjugated streptavidin according to the manufacturer’s instructions for the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific, Shanghai, China). For supershift analysis, 200 ng of anti-EZH2 antibody (Cell Signaling Technology, MA, USA) or IgG were added into the Protein–lncRNA binding reactions, followed by gel electrophoresis and ECL visualization.