Enhanced chemiluminescent substrate

Total proteins were extracted and quantified. Protein (30 μg) was separated in an SDS-PAGE gel and then electrotransferred onto a PVDF membrane (0.22 μm pore; Millipore, USA). Membranes were incubated with primary antibodies overnight at 4 °C with subsequent incubation with secondary antibodies labeled with horseradish peroxidase. The immunoblots were detected by an enhanced chemiluminescent substrate (Millipore) on the ChemiDoc MP system (Bio-rad, USA). Primary antibodies: BDNF (rabbit, 1/500, Abcam, Cambridge, UK); PAR-2 (mouse, 1:1000, Santa Cruz, USA), phospho-p38 (Thr180/Tyr182), p38, phospho-p65 (Ser536) and p65 antibodies are from Cell signaling Technology, USA (Rabbit, 1/1000); GAPDH (1:1000, Beyotime, Nanjing, China); horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibodies (1:10000, Zhongshan Gold Bridge, Beijing, China).

Total protein was extracted from prostate tissue and cell lines using radioimmunoprecipitation assay (RIPA) buffer, and protein concentrations were determined using the BCA Protein Reagent Kit (Beyotime, China) according to the manufacturer's instructions. Proteins (100 μg) were separated via SDS-PAGE on an 8% gel, transferred to a polyvinylidene fluoride membrane, and incubated with the following antibodies: rabbit anti-EphA5 (1:500, Abcam, CA) and mouse anti-beta-actin (1:1000, Santa Cruz, CA) overnight at 4°C. After washing, the membranes were incubated with HRP-conjugated goat polyclonal secondary antibodies to mouse IgG and rabbit IgG (1:5000, Abcam, CA) for 2 h and visualized with enhanced chemiluminescent substrate (Millipore, CA). Then, immunoreactive bands were quantified using the LAS-3000 system (Fuji Film, Japan).

Protein extracts were prepared from the tissues and cells using a RIPA lysis buffer supplemented with PMSF (Beyotime Institute of Biotechnology, Jiangsu, China), protein concentrations were measured using a bicinchoninic acid (BCA) Protein Assay kit (Beyotime). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, California, USA), and then transferred onto PVDF membranes (Millipore, Darmstadt, Germany). Following blocking with 5 % nonfat dry milk (Bio-Rad) at room temperature for 1 h, the membranes were immunoblotted overnight at 4 °C with primary antibodies against ACTN4 (1:3000, Proteintech), GCM-1(1:500, Proteintech), AKT (1:1000, Abcam), AKT phosphorylated at serine 473 (1:1000, Cell Signaling Technology, Danvers, MA, USA), p-GSK3β (1:1000, Cell Signaling Technology), Snail (1:1000, Cell Signaling Technology), N-cadherin (1:1000, Cell Signaling Technology), Vimentin (1:1000, Abcam), or β-actin (1:1000, Proteintech). After the membranes were rinsed with TBST, another incubation with the respective HRP-conjugated secondary antibodies (1:5000, Zhong San Golden Bridge Crop, Beijing, China) was carried out at room temperature for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescent substrate (Millipore), and the images were captured and analyzed using the ChemiDoc XRS + system (Bio-Rad).

Tissue and cellular protein samples were prepared using RIPA lysis buffer. The following antibodies were used: anti-LC3 (Bimake, China), anti-SQSTM1/P62 (Proteintech, China), anti-Beclin1 (Proteintech, China), anti-ATG5 (Wanleibio, China), anti-Bax (Servicebio, China), anti-BCL2 (Proteintech, China), anti-cleaved caspase-3 (Proteintech, China), GAPDH (Proteintech, China), anti-mTOR (Cell Signaling Technology, United States), anti-mTOR Ser(P)2448 (Cell Signaling Technology, United States), anti-AKT (Cell Signaling Technology, United States), and anti-p-AKT (Cell Signaling Technology, United States). The total isolated protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, United States), and the membranes were incubated overnight with a primary antibody at 4°C. The membranes were supplemented with secondary antibodies conjugated to horseradish peroxidase and subsequently detected with an enhanced chemiluminescent substrate (Millipore) and visualized via a Versa Doc imaging system (Bio-Rad, United States).