Molecular imager versadoc mp 5000 system

Prostate tissues and cells were washed by PBS and lysed in lysis buffer (Beyotime, Beijing, China). Then, the lysates were incubated on ice for 30 min and oscillated for 30 s. After centrifugation at 10 000 g for 30 min at 4°C, the supernatant was collected to measure the protein concentrations by BCA kit (Solarbio, Beijing, China). The proteins separated by the SDS-PAGE were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Then, the membranes were incubated overnight at 4°C for 1 h after blocking with primary anti-clusterin (1: 1000, Santa Cruz Biotechnology) and anti-β-actin (1: 1000, Santa Cruz Biotechnology). The membranes were then probed with anti-rabbit IgG (1: 50 000, Cell Signaling Technology, Beverly, MA, USA). The bands were determined by a Molecular Imager VersaDoc MP 5000 System (Bio-Rad, Hercules, CA) and densitometry was determined with Quantity One (Bio-Rad).

Total protein was extracted in ice-cold radioimmunoprecipitation assay buffer containing protease inhibitors (Beyotime Institute of Biotechnology), and quantified using a BCA protein assay kit. Proteins were denatured, separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane by the wet transfer method. The membranes were blocked with 5% skimmed milk in Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature and incubated with blocking solution containing primary antibody (1:400, anti-collagen III; 1:400, anti-MMP8; 1:400, anti-MMP14; 1:400, anti-TIMP2; 1:400, anti-CSE; 1:400, anti-TGF-β; 1:400, anti-TNF-α; 1:400, anti-NF-κB; 1:400, anti-STAT1/3/5/6; 1:400, anti-JAK-1/2; 1:1,000, anti-eIF2α; 1:1,000, anti-GRP94; 1:1,000, anti-caspase-3; and 1:1,000, anti-Bcl-2) overnight at 4°C. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000) for 1 h at room temperature. Next, the membranes were washed in TBST buffer three times and subjected to chemiluminescence detection assay. The bands were analyzed with a Molecular Imager VersaDoc MP 5000 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

RIPA buffer was used for the extraction of whole protein from both PC cell lines and tissue samples. A total of 20 µg extracted proteins were loaded per lane and separated by 10% SDS-polyacrylamide gels and transferred onto the polyvinylidene fluoride (PVDF) membrane. After blocked with 5% BSA (SABC, Shanghai, China) for 1 h at room temperature, the membranes were immunostained with the primary antibody overnight at 4°C and the secondary antibody for 1 h at room temperature. Equal amount of protein sample loading was monitored using an anti-β-actin antibody. Blots were detected by an enhanced chemiluminescence system (Thermo Fisher Scientific, Inc.) and images were captured with a Molecular Imager VersaDoc™ MP 5000 system (Bio-Rad Laboratories). The primary antibodies used and their dilutions are listed in Table II.

Liver tissues were washed by phosphate-buffered saline (PBS) and lysed. Then, the lysates were incubated on ice for 30 min and oscillated for 30 s. After centrifugation (10 000 g for 30 min at 4°C), the supernatant was obtained and the protein concentrations were determined by use of a bicinchoninic acid (BCA) kit. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the separated proteins were transferred onto polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, United Kingdom). After blocking for 1 h, the membranes were incubated overnight at 4°C with following primary antibodies: TLR4 (1: 100), TRAF6 (1: 500), and β-actin (1: 500). The membranes were then incubated with secondary antibodies (1: 3000). The bands were determined by use of the Molecular Imager VersaDoc MP 5000 System (Bio-Rad, Hercules, CA). The densitometry was determined with Quantity One software (Bio-Rad).