Radioimmunoprecipitation assay lysis buffer

The exosome pellet was dissolved in radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4°C, and the protein concentration was determined using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (17 (link),18 (link)). The proteins (20 µg/lane) were separated via 15% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% non-fat dried milk for 1 h, followed by incubation for 1 h with anti-CD9 (1:1,000; cat. no. 13174, Cell Signaling Technology, Inc.) and anti-heat shock protein 90α (HSP90α; 1:1,000; cat. no. 8165, Cell Signaling Technology, Inc.) primary antibodies, and subsequent incubation for 1 h with the secondary antibodies (horseradish peroxidase-conjugated anti-rabbit immunoglobulin G; 1:1,000; cat. no. 7074, Cell Signaling Technology, Inc.). The bands were visualized using the SuperSignal chemiluminescence system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All steps were performed at room temperature.

Western blotting was performed as described previously.14 (link) Briefly, cells were first washed twice with cold phosphate-buffered saline (PBS). Total proteins were extracted from the cells after incubating in radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with complete protease inhibitor cocktail (Hoffman-La Roche Ltd., Basel, Switzerland) for 1 hour on ice. The supernatants were collected after centrifugation at 15,000× g at 4°C for 20 minutes. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal amounts of denatured proteins were separated by electrophoresis on SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. After blocking nonspecific binding for 1 hour using 5% nonfat milk, the membranes were incubated overnight on ice with primary antibodies against β-actin (Sigma-Aldrich Co.) and RRAD, cyclin A2, cyclin B1, cyclin D1, cyclin E1, cyclin-dependent kinase (CDK) 2, CDK 4, CDK 6, Bax, PARP, caspase-3, GLTU1, GLUT4, LDHA, LDHB, FBP1, PKM2, and PGK1 (Abcam).

HUVECs (2×10⁵ cells/well) were seeded into 6-well plates 12 h prior to transfection. Ox-LDL was added to the cells 24 h following transfection and the cells were incubated for an additional 48 h. The cells were washed twice with PBS and lysed in radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc.), supplemented with protease inhibitors (Roche Diagnostics, Basel, Switzerland). The total protein concentrations were determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The protein lysates were separated on an 8–12% gradient gel by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat dry milk in PBS buffer for 1 h at room temperature, then incubated with rabbit anti-human LOX-1, Bcl-2, Bax, β-actin, and caspase-3 primary antibodies (dilutions, 1:1,000) overnight at 4°C. Subsequently, the membrane was incubated with the secondary antibody for 1 h at room temperature and the immunoblotting signal was developed using an Enhanced Chemiluminescence Western Blotting Analysis kit (Invitrogen; Thermo Fisher Scientific, Inc.) and exposed on X-ray films (Kodak, Rochester, NY, USA). The relative protein expression levels were normalized to β-actin and analyzed using ImageJ software (https://imagej.nih.gov/ij/).

The stimulated cancer cell lines were lysed with radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Danfoss, MA, USA) containing phenylmethanesulfonyl fluoride (Beyotime). Equal amounts of protein lysate were separated on 10% or 12.5% SDS PAGE gels (Abcam, Cambridge, UK) and then transferred to polyvinylidene fluoride membranes (EMD Millipore). The membranes were incubated with the primary antibodies at 4°C overnight. The following primary antibodies were used: anti-human matrix metallopeptidase 2 (MMP2) (#4022; Cell Signaling Technology), MMP9 (#13667; Cell Signaling Technology), p-ERK (4370P; Cell Signaling Technology), extracellular signal-regulated kinase 1/2 (ERK1/2) (4695P; Cell Signaling Technology), Akt (#4691; Cell Signaling Technology), p-Akt (#4060; Cell Signaling Technology), Bcl-2 (#4223; Cell Signaling Technology), Bax (#2772; Cell Signaling Technology), p27 (#2552; Cell Signaling Technology), p-mTOR (#2971; Cell Signaling Technology), mTOR (2983P; Cell Signaling Technology), cyclinD1 (#2978; Cell Signaling Technology), and GAPDH (AF0009; Beyotime). After incubation, the membranes were incubated with an horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Then, the membranes were washed six times with TBST (PBS with 0.05% Tween20). The blot bands were visualized by Find-do ×6 Tanon (Tanon, Shanghai, China).

Cellular lysates were prepared in chilled radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc.), and the protein concentrations were determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Proteins (10 µl) were loaded and separated by electrophoresis on 10% SDS-polyacrylamide gels at 110 V for 2 h. Following electrophoresis, the SDS-PAGE gels were transferred electronically to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). PVDF membranes were blocked using a solution containing 5% skimmed milk and incubated overnight at 4°C with the following antibodies: Anti-p62 and anti-β-actin (1:1,000). Following washing with Tris-buffered saline with Tween-20, the membranes were incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit immunoglobulin and monoclonal antibodies against β-actin. Reactive proteins were visualized using an Immobilon western horseradish peroxidase chemiluminescence kit (EMD Millipore, Billerica, MA, USA). Relative p62 expression levels as analyzed using ImageJ software version 1.47 (National Institutes of Health, Bethesda, MD, USA).

Protein samples were lysed from OC cells that underwent transfection, using a radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Protein samples were quantified with the Pierce BCA Protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) and were then boiled for 10 min in sodium dodecyl sulfate (SDS) sample buffer. Protein samples (20 µg) were separated with 10% SDS-PAGE, and subsequently transferred onto polyvinylidene membranes via electroblotting. Following blocking with 5% dried milk for 2 h at room temperature, membranes were then incubated with anti-PPP2R2A, anti-Cyclin D1 (cat. no. 2978; 1:1,000), anti-BAD, anti-phosphorylated (p)-Rb (cat. no. 8516; 1:1,000), anti-Rb (cat. no. 9313; 1:1,000) and anti-α-tubulin antibodies (cat. no. 2144; 1:1,000; all from Cell Signaling Technology, Inc.) overnight at 4°C, and then incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (cat. no. 7074; 1:5,000, Cell Signaling Technology, Inc.) for 2 h at room temperature. The protein bands were detection by an enhanced chemiluminescence western blotting substrate (Pierce; Thermo Fisher Scientific, Inc.) and quantified using ImageJ software version 1.48 (National Institutes of Health, Bethesda, MD, USA).