Enhanced chemiluminescence reagent

Every group cells were harvested and lysed in RIPA buffer (Cell Signaling Technologies Inc. Beverly, MA, USA). Bradford reagent (Bio-Rad, Hercules, CA, USA) was used to determine protein concentration. Then, adding 5ХSDS-loading buffer to give final concentration and heating the tubes at 100 degree C with locked capping for 5 min. The cell lysates (70 μg) were subjected to 12% SDS-PAGE. After electrophoresis, the cell extracts from SDS-PAGE were transferred to nitrocellulose membrane. Then, the membranes were incubated with rabbit dUTPase antibody (Trust Specialty Zeal, TST biological Trade Co., Ltd. USA) and β-actin antibody (Santa CruzBiotechnology, CA, USA)overnight at 4 °C. Furthermore, the membranes were incubated with HRP-conjugated antibodies for one hour. Visualization of the protein bands by using the enhanced chemiluminescence reagents (Invitrogen, Paisley, Scotland, UK). The bands analyzed by using the Image J 1.46r software (National Institutes of Health, Bethesda, MD, USA).

The prostate tissue from BPH patients was homogenized in cold RIPA buffer and centrifuged at 12,000 × g for 10 min at 4°C. Tissue lysates were then subjected to 10% SDS-PAGE and transferred to PVDF (Millipore, Shanghai, China). After blocking in Tris-buffered saline (TBS) buffer with 5% skim milk for 12 h, the membranes were incubated with antibodies against Ghrelin, Ghrelin receptor, Jak2, pJak2, Stat3 pStat3, and GAPDH at 4°C overnight. Then the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were detected using enhanced chemiluminescence reagents (Invitrogen, United States), and quantified by densitometric analysis using Image J software. The results were normalized to GAPDH to correct for loading.

Cells were seeded into 6‐well plates at a density of 5 × 10⁵ cells/well for 48 hours. The cells were then washed in PBS and lysed in ice‐cold lysis buffer. The protein contents of the lysates were determined, and equal amounts of proteins were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Bio‐Rad). Next, non‐specific interactions were blocked by incubation with 5% non‐fat milk‐tris‐buffered saline with 0.1% Tween‐20 at 37°C for 1 hour. The membranes were incubated with specific antibodies against EZH2 (1:4000; Proteintech) and β‐actin (1:2,000; Sigma) at 4°C for 12 hours to detect the corresponding proteins and then were washed and incubated with horseradish peroxidase‐conjugated secondary antibody (Bioss Antibodies) for 1 hour at 37°C. To analyse relative protein expression levels, immunoblots were detected with enhanced chemiluminescence reagents (Invitrogen Life Technologies) and exposed to X‐ray films. Quantity One software (Bio‐Rad) was used, with β‐actin as the internal reference.

All groups of cells were harvested and lysed in RIPA buffer (Cell Signaling Technologies Inc. Beverly, MA, USA). Bradford reagent (Bio-Rad Laboratories Inc, Hercules, CA, USA) was used to determine protein concentration. Cell lysates were mixed with 5ХSDS-loading buffer (4:1, v/v) and heated the tubes at 100 °C with locked capping for 5 min. Samples (40 μg of protein) were subjected to 10% SDS-PAGE. After electrophoresis, the cell extracts from SDS-PAGE were transferred to polyvinylidene fluoride transfer membrane (Bio Trace^TM PVDF, 0.45 μM, Pall Life Sciences Corp., Mexico). Membranes were then incubated with anti-RRM1 (D12F12) antibody (Cell Signaling Technology, Inc., MA, USA), anti-RRM2 (EPR11820) antibody, anti-p53R2 (EPR8816) antibody (Abcam Ltd, Cambridge, UK) and β-tubulin antibody (Santa Cruz Biotechnology, CA, USA) overnight at 4 °C, respectively. The membranes were further incubated with HRP-conjugated antibodies for one hour. The protein bands were visualized using the enhanced chemiluminescence reagents (Invitrogen Corp., Paisley, Scotland, UK), and were analyzed with the Image J 1.46r software (National Institutes of Health, Bethesda, MD, USA).

Following the experiment, mice were euthanized with an overdose of anesthetic combined with carbon dioxide. Tumors were quickly obtained from the mice, then coarsely minced and homogenized. Western blotting was then performed as previously described [98 (link),99 (link),100 (link)]. For detection, we used antibodies against β-actin, Bcl-2, and COX-2 from Sigma-Aldrich Inc. (St. Louis, MO, USA); VEGF and EGFR were also purchased from Sigma-Aldrich (St. Louis, MO, USA); and cleaved caspase 3, phosphorylated ERK (threonine 202/tyrosine 204), and ERK were from Cell Signaling Technology (Beverly, MA, USA). Enhanced chemiluminescence reagents (Thermo Scientific, Rockford, MA, USA) generated the immunoreactive signals and UVP ChemStudio (Analytik Jena, Upland, CA, USA) was used for signal detection. The quantification of protein expression and phosphorylation was performed using ImageJ software from the National Institutes of Health (NIH; Bethesda, MA, USA).