P β catenin

Cells were harvested and lysed in RIPA lysis buffer (Thermo Fisher Scientific, USA) for extracting total proteins. The protein concentrations were measured by a BCA protein assay kit (Transgen Biotech, China), separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. The membranes were then blocked by 10% nonfat milk and incubated with the corresponding antibody (CKIP-1, Akt, p-Akt, Gsk3β, β-catenin, p-β-catenin, Smurf1, and GAPDH; Abcam, Cambridge, MA, USA) diluted according to the instructions for 12 h at 4°C. After washing 3 times with PBS/0.1% Tween-20, membranes were incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG and anti-rabbit IgG at 1:10000 dilution; Abcam). Protein expression levels were detected with ECL detection solution (Millipore, USA) and visualized with chemiluminescence detection system. The bands signal were analyzed using a gel imaging system (Bio-Rad).

Cells were grown on glass coverslips until adhesion, fixed with 4 % formaldehyde in PBS for 10 min, and permeabilized with 0.2% Triton X-100/PBS at room temperature. As for paraffin-embedded specimens (4μm), the sections were rehydrated by xylene, subjected to antigen retrieval by irradiating in target retrieval solution (TRS, DAKO, USA) with microwave oven. The sections were blocked by 5% bovine serum albumin (Sigma, USA) and then incubated overnight at 4°C with LC-3B, p38, p-p38, β-catenin and p-β-catenin (Abcam, Cambridge, UK). They were then washed with PBS and incubated with Alexa Fluor 488 IgG (Invitrogen, USA). Nuclei were stained with 1 μg/mL DAPI (Sigma, USA) in the dark. Finally, coverslips were mounted with SlowFade^® Gold antifade reagent (invitrogen, USA) and visualized in a laser confocal scanning microscope (Leica, Germany).

The following chemicals were used: ADAM10 inhibitor (GI254023X; #SML0789, Sigma-Aldrich, Taufkirchen, Germany); ADAM10/17 inhibitor (GW280264X; #AOB3632, Aobious Inc., Hopkinton, MA, USA); human VEGF-A (#V4512, Sigma-Aldrich); TNFα (#H8916, Sigma-Aldrich); protease activator APMA (P-aminophenylmercuric acetate; #A9563, Sigma-Aldrich); γ-secretase inhibitor (flurbiprofen [(R)-251,543.40–9]; #BG0610, BioTrend, Cologne, Germany).
For Western blotting, primary antibodies reactive with the following antigens were used: P-β-catenin (Tyr142; diluted 1:500; #ab27798, abcam, Cambridge, UK); P-VEGF-R2 (Tyr1214; 1:1000, #AF1766, R&D Systems, Wiesbaden, Germany); VE-cadherin (BV9; 1:500; #sc-52,751, Santa Cruz Biotechnology, Heidelberg, Germany); VE-cadherin (1:1000; #2158S); ADAM10 (1:500–1:1000; #14194S); ADAM17 (1:1000; #3976S), β-catenin (1:1000; #9587S); VEGF-R2 (1:1000; #9698S); P-VEGF-R2 (Tyr1175; 1:1000; #2478S, all from Cell Signaling Technology, Frankfurt, Germany); and β-actin-POD (1:25,000; #A3854, Sigma-Aldrich). HRP-conjugated secondary antibodies were from Cell Signaling Technology.
For immunofluorescence microscopy, the following antibodies were used: anti-VE-cadherin (1:50; #2158S); anti-mouse IgG (H + L), Alexa Fluor 555 conjugate (1:1500; #4409); and anti-rabbit IgG (H + L), Alexa Fluor 488 conjugate (1:1500; #4412) (all from Cell Signaling Technology).

The total protein was extracted from human PCa cells following the instructions of the total protein extraction kit (bl521a; Biosharp, Shandong, China); subsequently, the protein concentration was detected using the diquinoline formic acid method. The denatured protein samples were separated by electrophoresis according to which membrane was transferred using the semi-dry method. After sealing the membrane with skimmed milk powder for 2 h, we added β-actin (gb12001; Servicebio, Wuhan, China), Wnt3a (2721; Cell Signaling Technology, Danvers, MA, USA), GSK3 β (ab2602; Abcam, UK), phosphorylated (p)-GSK3 β Ser9 (ab131097; Abcam, UK), β-catenin (ab32572; Abcam, UK), p- β-catenin (ab27798; Abcam, UK), C-myc (ab32072; Abcam, UK), Cyclin D1 (2978; Cell Signaling Technology, USA), vimentin (3195; Cell Signaling Technology, USA), E-cadherin (60330-I-Ig, Proteintech, USA), and GPX2 (ab140130; Abcam, UK) antibodies. Subsequently, the membrane was incubated overnight at 4 °C, then incubated with primary and secondary antibodies at room temperature for 2 h, exposed, and developed using the ECL film. The protein expression was analyzed, and the experiment was repeated three times.