Ab186006

The binding condition of MALAT1 with EZH2 was detected using a Magna RIP kit (Millipore, Billerica, MA, USA). The cells reaching 90% confluence were lysed in RIP lysis buffer, and subsequently the supernatant was collected, which was then divided into two equal parts. Next, 100 μL of cell extraction was incubated with 900 μL of RIP buffer containing the beads conjugated with EZH2 antibody (ab186006, 1:1,000, Abcam, Cambridge, MA, USA) or immunoglobulin G (IgG) at 4°C overnight. After instantaneous centrifugation, the centrifugal tube was placed on the magnetic separator and the supernatant was discarded. Afterward, the beads were washed with 500 μL of RIP Wash Buffer six times. The samples were incubated with protease K at 55°C for 30 min and shaken continuously to detach protein. TRIzol-chloroform was used to isolate the immunoprecipitated RNA, and the enrichment of MALAT1 was evaluated by qRT-PCR.¹⁸ (link)

Proteins were extracted from cells using Radio‐Immunoprecipitation Assay buffer (RIPA, Beyotime Institute of Biotechnology), and the concentration of proteins was calculated by a BCA protein assay kit (Beyotime Institute of Biotechnology). An equal amount of proteins (20 μg/lane) was subjected to 12% SDS‐PAGE, followed by an electrophoretic transfer onto a polyvinylidene fluoride membrane. After being blocked with 5% non‐fat milk dissolved in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 hour at room temperature, membranes were incubated with rabbit polyclonal antibodies against EZH2 (ab186006, 1:1,000, Abcam) or β‐actin (ab8227, 1:2,000, Abcam) overnight at 4°C. The next morning, membranes were washed with TBST three times and probed with horseradish peroxidase‐conjugated (HRP) goat anti‐rabbit IgG H & L secondary antibody (ab6721, 1:10,000, Abcam) for 1 hour at 37°C. After being washed with TBST, immunoreactive bands were developed by enhanced chemiluminescence (Beyotime Institute of Biotechnology) and the grey values of each blot were calculated by ImageJ version 1.50 (National Institutes of Health). The relative expression of the target protein was calculated by normalization to β‐actin.

Cells were transfected with biotinylated LINC00152-Sense strand and LINC00152-Antisense strand (50 nM for each). At 48 h post transfection, cells were washed, vortexed and incubated with specific cell lysis buffer (Ambion, Austin, TX, USA) for 10 min, taking a 50 mL portion of cell lysate as a control. The remaining lysate was incubated with M-280 streptavidin magnetic beads (Sigma, St. Louis, MO, USA) pre-coated with RNase-free and yeast tRNA (Sigma, St. Louis, MO, USA) for 3 h at 4 °C, followed by two washes with cold lysis buffer, three washes with low salt buffer, and one wash with high salt buffer. Afterwards, total protein was extracted with high-efficiency RIPA lysis buffer and then the expression of EZH2 (1:100, ab186006, Abcam, Cambridge, UK) was determined by Western blot analysis. The experiment was repeated in triplicate.

The total protein content was extracted using the RIPA lysis buffer (Beyotime) with protease inhibitors, followed by protein concentration quantitation using a BCA kit (20201ES76, Yeason, Shanghai, China). Protein was separated by PAGE and then transferred onto PVDF membrane by a wet transfer. Following blocking with 5% BSA, membranes were probed with the primary antibodies against SOX5 (ab94396, Abcam, 1:1000), EZH2 (ab186006, Abcam, 1:2000), osteocalcin (OCN; ab133612, Abcam, 1:1000), Runx2 (ab236639, Abcam, 1:1000), Collagen I (ab34710, Abcam, 1:2000), GAPDH (ab8245, Abcam, 1:3000; internal reference) overnight at 4 °C. The membrane was then re-probed with diluted secondary antibodies against HRP-labeled goat anti-rabbit IgG (ab6721, Abcam) or goat anti-mouse IgG (ab6789, Abcam) for 1 h at room temperature. Following visualization in chemiluminescence reagent, protein quantitative analysis was conducted by the ImageJ software.

Equal amounts of total protein (approximate 80  µg) were separated by a 12% SDS-polyacrylamide gel and transferred onto 0.2-µm PVDF membranes (Roche, Basel, Switzerland). The blots were incubated overnight at 4 ° C with antibodies (anti-EZH2: 1:5000, ab186006, Abcam, CA, USA; anti-Collagen I: 1:1000, ab6308, Abcam; anti- α-SMA: 1:1000, ab19245, CST; anti-SOCS7: 1:1000, ab224589, Abcam; anti- β-actin: 1:2000, ab8227, Abcam) and were subsequently incubated with a secondary antibody (1:5000, ab205718, Abcam). The protein signals were detected with ECL (Pierce Biotechnology, Rockford, IL) and digitized and analyzed densitometrically using ImageJ software.

Tumors were removed from the euthanized nude mice, dried, fixed in 4% paraformaldehyde solution, routinely paraffin-embedded, sectioned into 4 μm serial sections, and subjected to antigen retrieval. The sections were rinsed with PBS and blocked by normal goat serum. HistostainTMSP-9000 immunohistochemical staining kit (Zymed) was used to stain the sections. The sections were incubated with indicated primary antibodies at 4°C overnight. After rinsing with PBS, sections were incubated with secondary antibody goat anti-rabbit (ab6721, 1:1000, Abcam) and goat anti-mouse (ab6728, 1:1000, Abcam). The results were visualized after addition of diaminobenzidine for 5 to 10 min, with the staining time adjusted by monitoring under the microscope. After re-staining with hematoxylin for 1 min, the sections were mounted and photographed. Five representative high-frequency fields of view (NIKON, Japan) were selected for observation and counting, with the cytoplasm being positive if brown or yellow colored. The antibodies used were: SETDB1 mouse antibody (ab107225, 1:100, Abcam), EZH2 rabbit antibody (ab186006, 1:100, Abcam), AKT rabbit antibody (ab179463, 1:250, Abcam), PT308-AKT rabbit antibody (ab38449, 1:100, Abcam), PS473-AKT rabbit antibody (ab81283, 1:100, Abcam), and AKT K140me3 rabbit antibody (1:1000, CST).