S8820

Three intact adults or five flies per biological sample collected during development, depending on the driver being used, were homogenized in a 200 µL of NETN buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40) that was supplemented with a protease inhibitor (PI; S-8820, Sigma-Aldrich). The resulting lysates were then diluted with 200 µL 0.5% SDS in PBS. Diluted lysates were sonicated, centrifuged at 4500× g for 1 min at room temperature and then diluted further by combining 100 µL lysate with 400 µL PBS. A total of 35 µL of this final lysate of each sample were added to a Bio-Dot apparatus (Bio-Rad) and filter-vacuumed through a 0.45 µm nitrocellulose membrane (Schleicher and Schuell, Keene, New Hampshire) that had been pre-incubated with 0.1% SDS in PBS. After samples were filter-vacuumed through the membrane, the membrane was rinsed twice with 0.1% SDS in PBS, incubated in primary and then secondary antibody, and analyzed via Western blotting.

GST-fusion proteins were expressed in BL21 E.coli strain and purified as described previously⁶⁸ . Expression level and purity of these proteins was evaluated based on coomassie brilliant blue-stained gel (Fig. S3). Crude synaptosome whole brain fraction was lysed in buffer containing 1% Triton, 130 mM NaCl, 50 mM Tris, pH 7.2, protease (Sigma, #S8820) and phosphatase (Sigma, #P0044) inhibitors. Protein extracts were incubated overnight at 4 °C with the corresponding GST-fusion proteins immobilized on glutathion-agarose beads (Sigma, #G4510). Then beads were washed four times with the 0.1% Triton X-100, 130 mM NaCl, 50 mM Tris, pH 7.2, buffer and boiled in sample buffer for 7 minutes at 95 °C. After boiling supernatant was collected, separated by SDS-PAGE and probed with the anti-EB3 antibody (1:2000, Sigma, #SAB4200606).

Previous studies^{65 (link)} were used to guide the western blot. To reduce tissue proteases and facilitate protein extraction, adhesion tissue samples were first homogenized in RIPA lysis buffer solution (Sigma-Aldrich S8820, USA). Then, a loading buffer was used to dilute the protein samples, which were then heated in a 95 °C bath for five minutes. Then, the isolated proteins were electrophoresed on a polyvinylidene fluoride membrane (PVDF) at 100 V for 1–2 h using sodium dodecyl sulphate–polyacrylamide (SDS-PAGE, 120 V). PVDF membrane was treated in a particular buffer (5 percent non-fat milk) for a night to suppress endogenous peroxidases. Hence, the membranes were washed in Tris-buffered saline (pH = 7.2, including 0.1 percent Tween 20, × 3, 15 min each time) and incubated with TNF-α, TGF-β1, VEGF, and β-Actin (E-AB-40015, E-AB-67255, E-AB-33090, E-AB-20058) antibodies for two hours at 4 °C. Unattached antibodies were rinsed with a washing solution before being incubated for 60 min at 25 °C with horseradish peroxidase (HRP)-conjugated secondary antibody (E-AB-1003, E-AB-1001). Finally, an enhanced chemiluminescence detection kit (ECL, Thermo Scientific, USA) was used to visualize the blots. Using improved laser densitometer software, the relative intensity of protein/β-Actin was determined (Arash-Teb-Pishro, Iran).

Total RNA was isolated using the mirVana miRNA Isolation Kit (Invitrogen, AM1560) following the manufacturer’s instructions. After RNA extraction, RNA samples were reverse-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368813). Real time quantifications of TMPRSS2-ERG fusion mRNA was performed with specific TaqMan gene expression assay (Assay ID: Hs03063375_ft). Real-time PCR data were normalized to the endogenous control β-actin. The relative fold changes of candidate genes were analyzed by using 2^–ΔΔCT method.
Protein extraction and immunoblot analysis were performed using the standard protocol. In brief, cells were lysed in RIPA buffer supplemented with protease/phosphatase inhibitors (Sigma, P5726 and S8820, respectively). Samples containing 10μg protein were electrophoresed on a 4–12% Tris-Glycine gel. The separated proteins were electro-transferred to a nitrocellulose membrane (Bio-Rad, 1620112) for western blot analysis. All primary antibodies were used at 1:1000 dilution. The band intensities representing different protein expression levels were quantitated with reference to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control bands. The intensities of protein bands were quantitated using ImageJ Gel Analysis program.