Anti caspase3

To determine the effect of CW-33 on JEV-induced apoptosis and signal pathway activation, TE671 cells were simultaneously infected with JEV at an MOI of 0.05 and treated with/without various concentrations of CW-33. After a 36-h incubation, the lysate of mock, infected, infected/treated, or treated cells was mixed with the SDS-PAGE sample buffer and heated for 10 min. Proteins in the lysates were resolved by SDS-PAGE and transferred to the nitrocellulose membrane. Resultant membranes were blocked with 5% skim milk, then reacted with appropriately diluted antibodies, including antibodies anti-caspase 3 (IMGENEX, San Diego, CA, USA), anti-phospho-Akt Ser473, anti-phospho-mTOR Ser2248, anti-phospho-JAK1 Tyr1034/Tyr1035, anti-phospho-JAK2 Tyr1007/Tyr1008, anti-phospho-Tyk2 Tyr1054/Tyr1055, anti-phospho-STAT1 Ser727 (Cell Signaling, Danvers, MA, USA), and anti-beta actin (NOVUS BIOLOGICALS, Littleton, CO, USA). Immune complexes were detected with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibodies, followed by enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Soluble human TRAIL was obtained from Enzo Life Sciences (Loerrach, Germany). Staurosporine (STS) and the JNK inhibitor SP600125 were purchased from Sigma (Deisenhofen, Germany). GW280264X was kindly provided by GlaxoSmithKline (Stevenage, UK). The antibodies used in this study were obtained as follows: anti-Caspase-3 (Imgenex, San Diego, CA, USA); anti-Caspase-8, anti-PARP (BD Pharmingen, Franklin Lakes, NJ, USA); anti-GAPDH, anti-DR4, anti-Notch1, anti-Sp1 (Santa Cruz Biotechnology, San Diego, CA, USA); anti-Integrin β4 (ITGB4), anti-pJNK, anti-cJun, anti-phospho-cJun (Cell Signaling, Danvers, MA, USA); and anti-DR5 (Sigma).

Immunoblotting was performed using anti-phospho-RB (Cell Signaling, Danvers, MA, USA), anti-p62 (GeneTex, Irvine, CA, USA), anti-LC3B (Novus biologicals, Littleton, CO, USA), anti-caspase3 (Novus biologicals), anti-γH2AX (Cell Signaling), anti-PIK3C3 (GeneTex) and anti-GAPDH (GeneTex) antibodies, followed by visualization using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence detection system.

The cell lysates were prepared from PCa cells treated with GTEE or vehicle (0.3% ethanol) using PRO-PREP Protein Extraction Solution (iNtRON technology, South Korea) with protease inhibitors added. The protein concentrations of the cell lysates were assayed using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). For Western blotting, 50 µg of protein extract was loaded into the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking by 5% non-fat milk in PBST (PBS with Tween-20) buffer for 1 h at room temperature, PVDF membranes were incubated with primary antibodies for overnight at 4 °C, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody. The reactive signals were visualized using an Enhanced Chemiluminescence Kit (Amersham Biosciences, Arlington Heights, IL, USA). Subsequently, the reactive protein bands were scanned and quantified using ImageJ software. Primary antibodies were used as follows: anti-SREBP-1, anti-FASN, anti-AR (Santa Cruz Biotechnology, Dallas, TX, USA), anti-SREBP-2 (Abcam, Cambridge, MA, USA), anti-caspase 3 (Novus Biologicals, Littleton, CO, USA), anti-PARP (GeneTex, Irvine, CA, USA), and anti-β-actin (Millipore, Burlington, MA, USA).

The protein levels in the penile tissues were measured by Western blotting. After the penis tissue was taken out, it was fully ground in liquid nitrogen. It was then lysed with radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific), and the protein concentrations were detected by a bicinchoninic acid protein detection kit. In 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, equal amounts of protein were injected, electrophoresed, and transferred to polyvinylidene fluoride membranes. These membranes were blocked with 5% skimmed milk for 2 hours and incubated overnight at 4 °C with the following primary antibodies: anti–neuronal nitric oxide synthase (anti-nNOS, 1:1000; Abcam), anti-GRP78 (1:1000; Abcam), anti-CHOP (1:1000; CST), anti-PERK (1:1000; CST), anti–p-PERK (1:1000; CST), anti–caspase 3 (1:1000; Novus Biologicals), anti–α-SMA (1:1000; CST), and anti-OPN (1:1000; Novus Biologicals). After the membranes were washed 3 times, they were incubated with secondary antibody at room temperature for 1 hour. An enhanced chemiluminescence system (SH-Compact 523; SHST) was used to detect the protein bands, which were then quantitatively analyzed with ImageJ (version 1.52a; National Institutes of Health). The protein levels were normalized to the levels of β-actin or GAPDH (1:1000; CST).

Total protein samples were extracted from vehicle- or DFE (20 µg/mL)-treated PCa cells by PRO-PREP Protein Extraction Solution (iNtRON technology, Seoul, South Korea) adding with protease inhibitors. A Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific) was utilized to determine the concentrations of proteins. The equal amounts of protein samples were loaded into SDS-PAGE gels for electrophoresis and then proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the blotted membranes were blocked using 5% non-fat milk in PBS with Tween-20. After blocking, the membranes were incubated with primary antibodies for overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Primary antibodies were used in this study as follow: anti-AR, anti-SREBP-1, anti-FASN (Santa Cruz Biotechnology, Dallas, TX, USA), anti-SREBP-2 (abcam, Cambridge, MA, USA), anti-caspase-3 (Novus Biologicals, Littleton, CO, USA), anti-PARP (GeneTex, Irvine, CA, USA), and anti-β-actin (Millipore, Burlington, MA, USA). The protein signals were visualized using an Enhanced Chemiluminescence Kit (Amersham Biosciences, Arlington Heights, IL, USA) and an imaging system (ImageQuant LAS 4000; GE Healthcare, Pittsburgh, PA, USA). The ImageJ software was utilized to quantify specific protein bands by normalized a loading control, β-actin.

Total protein samples were prepared from LNCaP and C4-2 cells treated with AIF or control by RIPA Lysis Buffer containing protease inhibitors. The amounts of proteins were determined by a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). The equal amounts of the protein samples were loaded in SDS-PAGE gel for electrophoresis [26 (link)]. After electrophoresis, the separated proteins were transferred into polyvinylidene difluoride (PVDF) membranes and blocked by 5% reconstituted skim milk in PBS with Tween-20. Subsequently, the blotted membranes were incubated with primary antibodies for overnight. The detailed information of the used primary antibodies in this study as follow: anti-AR, anti-FASN (Santa Cruz Biotechnology, Dallas, TX, USA), anti-HMGCR (Abcam, Cambridge, UK), anti-BAX (Cell Signaling Technology, Danvers, MA, USA), anti-caspase-3 (Novus Biologicals, Centennial, CO, USA), anti-PARP, and anti-β-actin (GeneTex, Irvin, CA, USA). Next, these membranes were incubated with the secondary antibody. The visualization of the specific protein signal was detected by Enhanced Chemiluminescence Reagent kit (Amersham Biosciences, Arlington Heights, IL, USA) with the ImageQuantTM LAS 4000 system. Finally, the quantification of the protein band was analyzed by an ImageJ software (ij153-win-java8) with normalizing β-actin protein.