Radioimmunoprecipitation assay lysis buffer

Total protein was isolated and collected from HGSOC cell lines with radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotech, Santa Cruz, CA, USA). The isolated proteins with equal quantity were further separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE; 10% or 12%), followed by transferring onto separate polyvinylidene fluoride membranes (Beijing Solarbio Science& Technology, Beijing, China). The antibodies used for immunoblotting were the following: GAPDH (Cell Signaling Technology, Beverly, MA, USA), SOCS7 (ab224589; Abcam, Cambridge, MA, USA), HuR (ab238528; Abcam), Cyclin D1 (ab16663; Abcam), Survivin (ab76424; Abcam), CDC25B (ab124819; Abcam), and FOXM1 (ab207298; Abcam). After incubating with horseradish peroxidase-conjugated secondary antibody (Beyotime Institute of Biotechnology), the signals were further examined based on an enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA).

Dot blot assays were used to identify proteins transported by EVs. Isolated EVs were lysed using radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology). EV lysates (4 μl) were spotted onto nitrocellulose blotting membrane and dried at RT for 10 min. Blocking of the membrane was performed in TBS-T buffer (2.4 g Tris base, 8.8 g NaCl, 1 mL Tween 20 [Sigma-Aldrich] diluted in 1 liter of distilled water [pH 7.6], added milk powder [3%], BSA [1%], Tween 20 [0.1%]) and was mixed for 1 h at RT using the rotating roller mixer SRT1 (Stuart Scientific). Afterwards the membrane was incubated with monoclonal antibody (MAb) CD11b (BioLegend), MAb CD35 (BioLegend), MAb CD9 (Thermo Fisher Scientific), and MAb CD63 (Merck) in blocking buffer overnight at 4°C using the rotating roller mixer. The membrane was then washed 3 times in TBS-T buffer for 10 min and incubated with the horseradish peroxidase-conjugated secondary antibody (Dako) at RT for 1 h using the rotating roller mixer SRT1. The membrane was washed 3 times in TBS-T buffer for 10 min each and subsequently incubated with an enhanced chemiluminescence (ECL) mix for 30 s. ECL mix was combined of solution A (luminol and enhancer solution) and B (peroxide solution) from Cheluminate-HRP PicoDetect (AppliChem). Signals from the nitrocellulose blotting membrane were assayed in Fusion FX imaging system (Vilber).

Proteins were extracted with radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology). Proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane that was probed with primary antibodies against B cell lymphoma (Bcl)-2 (1:400), Bax (1:200), LC3-1(1:400) and P62 (1:400), (all from Abcam); HO-1 (1:200), NQO1 (1:200), and UGT1A1 (1:200) (all from Santa Cruz Biotechnology Inc); and cleaved caspase-3 (1:200), Beclin-1 (1:200), ATG-3 (1:400), and ATG-7 (1:400; all from Cell Signaling Technology), followed by incubation with appropriate secondary antibodies. Immunoreactivity was visualized with the ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Gray value analysis was conducted with the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or laminin B1 (1:3000, Cell Signaling Technology).

NRVMs were homogenized on ice in radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Dallas, TX) containing the protease inhibitors leupeptin (5 µg/mL), aprotinin (2 µg/mL), and phenylmethylsulphonyl fluoride (PMSF; 1 mM). Lysates were centrifuged at 12,000g for 20 min at 4 °C and the supernatants were collected. The protein concentrations were determined, and aliquots containing 50 mg of protein were separated by electrophoresis on 4–12% Blot Bis–Tris Gels (Invitrogen Japan) in a NuPAGE MOPS SDS Running Buffer system (Invitrogen Japan), and sequentially transferred onto nitrocellulose membranes, according to the manufacturer’s instructions. Proteins were blotted using iBind Western Systems (Thermo Fisher Scientific) and detected by chemiluminescence (#34096, Thermo Fisher Scientific; #NEL113001EA, PerkinElmer, Inc., Waltham, MA, USA; ImageQuant LAS500, GE Healthcare UK Ltd., Buckinghamshire, England). The following primary antibodies used in this study were: anti-Bax (#14796), anti-caspase3 (#14220), anti-Cleaved caspase3 (#9661; All of them; Cell Signaling Technology), anti-SESN1 (ab134091; Abcam, Cambridge, MA, USA), anti-p-mTOR (S2448), anti-mTOR (7C10), anti-LC3B (#2775), and anti-β-actin (All of them; Cell Signaling Technology).