Proteinase inhibitor cocktail

Protein was extracted from cultured cells using RIPA-lysis buffer (50mM Tris-HCl pH8, 150mM NaCl, 1mM EDTA, 0.1% SDS, 0.5% Deoxycholate, 1% NP-40), supplemented with proteinase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). The protein extracts were quantified using the DC protein assay (BioRad, Hercules, CA, USA). For immunoblot analysis, 70μg of protein was resolved by SDS PAGE 4–12% Bis-Tris gels (Life Technologies) and blotted to PVDF membrane (BioRad) using NuPAGE reagents and equipment (Life Technologies, Carlsbad, CA, USA). Blots were blocked in 5% BSA PBS-Tween buffer for 1h. Membranes were then incubated with primary antibodies recognizing androgen receptor-D6F11 (#5153, Cell Signaling, Danvers, MA, USA), γH2Ax-Ser139 (sc:101696, Santa Cruz Biotechnology, Dallas, TX, USA), or fibrillarin-H140 (sc:25397, Santa Cruz) overnight at 4°C. Blots were then washed and incubated with goat anti-rabbit IgG-HRP as secondary antibody for detection (Santa Cruz). Protein bands were then visualized using Super Signal WestFemto Reagent Substrate (Thermo Scientific) and developed on the AlphaInnotech FluorChem 8900 system (ProteinSimple, San Jose, CA, USA). Densitometry of each specific protein band was determined using AlphaInnotech FluorChem 8900 software and normalized to its corresponding fibrillarin band.

One mL of human plasma was incubated with 0.5 mL of Invitrogen total exosome isolation reagent (Thermo Fisher Scientific) overnight at 4°C, then centrifuged at 10,000 x g for 1h at 4°C. Pellets were resuspended in 2 volumes of radioimmunoprecipitation assay buffer (RIPA, 150 μL, 50 mM Tris-HCI pH 7.4, 1% NP40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCI, 2mM EDTA), homogenized and sonicated. Thirty μg of protein was used for western blot analyses. Cell media from 6-OHDA- or vehicle-treated SH-SY5Y cells were collected after 8 h of incubation and centrifuged at 800 x g for 10 min at 4°C. Supernatants were filtered using a 0.22 μm filter and centrifuged at 100,000 x g for 90 min at 4°C. Pellets were resuspended in 0.5 mL PBS containing a proteinase inhibitor cocktail (Thermo Fisher Scientific) and centrifuged again at 100,000 x g for 90 min at 4°C. Supernatants were collected and 30 μg of protein were used for western blot analyses to verify exosome markers (CD63 and Alix).

The tumor tissues were homogenized by bead beating in RIPA buffer containing with 1% proteinase inhibitor cocktail (200 μL/20 mg tissue, ThermoFisher, #78429) within 10 min at 4 °C. The lysates were then centrifuged at 15,000 r.p.m. for 20 min at 4 °C, the supernatants were collected and quantified by BCA assay. Samples were dissolved in SDS-PAGE protein loading buffer (5X, Boster, AR1112) and boiled for 10 min, and equal amounts of each sample were applied to 12% Tris-glycine gel (Novex gel, Invitrogen) and subjected to electrophoresis, which was subsequently transferred to a PDVF (polyvinylidene difluoride) membrane and blocked in 5% non-fat milk blocking buffer for 1 h at room temperature. The membranes were incubation with the primary antibody overnight at 4 °C (STING (D1V5L) mAb, #50494, dilution 1/1000; Phospho-STING (Ser365) (D8F4W) mAb, #72971, dilution 1/1000; IRF-3 (D83B9) mAb, #4302, dilution 1/1000; Phospho-IRF-3 (Ser396) (D6O1M) mAb, #29047, dilution 1/1000, Cell Signaling; β-Actin, ab179467, dilution 1/1000, Abcam), and then horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, HRP-linked antibody, #7074, dilution 1/3000; Cell Signaling). Finally, the membranes were soaked in ECL substrate (Thermo Scientific) and imaged under Azure Biosystems software (v. 1.5.0.0518).