Anti cd81 antibody

Proteins were prepared as we previously documented (27 (link)). Total proteins were extracted from cells with RIPA lysis buffer mixed with phenylmethyl-sulfonyl fluoride (PMSF) and phosphatase inhibitor cocktail I (MedChemExpress, China). An equal amount of protein was separated on 10% or 12% SDS-PAGE gels based upon the molecular weight of the target protein and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies (anti-CD9 antibody, abcam, USA, #ab92726; anti-CD63 antibody, abcam, USA, #ab213090; anti-CD81 antibody, abcam, USA, #ab109201; anti-TSG101 antibody, abcam, USA, #ab83; anti-p53 antibody, abcam, USA, #ab179477; anti-PTEN antibody, Cell Signaling Technology, USA, #9559S; anti-mTOR antibody, Cell Signaling Technology, USA, #2983T; anti-phospho mTOR antibody, Cell Signaling Technology, USA, #5536T; anti-GAPDH antibody, SIGMA, USA, #G9545). Subsequently, proteins were detected by an enhanced chemiluminescence (ECL) system reagent (KeyGEN BioTECH, China) after incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein expression was calculated with Image J software and normalized to GAPDH expression.

Sodium dodecyl sulfate (SDS) sample buffer (0.5 M Tris-HCl, pH 6.8, 20% sucrose, 10% SDS, 1% bromophenol blue) was added to the concentrated samples. After equal amounts of protein (5 μg/lane) were separated on 4–20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA) under nonreducing conditions, the gels were blotted onto nitrocellulose membranes (Pierce, Waltham, MA, USA) in an electrophoretic transfer cell (Bio-Rad). Blots were blocked with 5% non-fat milk at 4 °C overnight and then probed with Anti-Collagen I antibody (1:1000; Abcam, Cambridge, UK, ab23446), anti-CD63 antibody (1:1000; Abcam, ab59479), anti-Hsp70 antibody (1:1000; Abcam, ab2787) and anti-CD81 antibody (1:1000; Abcam, ab59477) at room temperature for 1 h. After three washes with PBS-T (pH 7.4 and 0.1% Triton), the membrane was incubated with sheep anti-mouse IgG conjugated to HRP (NeoBioscience, Shenzhen, China) for 1 h at room temperature, followed by three washes in PBS-T. The band density was determined by Bio-Rad Quality One Software.

Cells and EVs were lysed in RIPA lysis buffer (Beyotime, China), and protein content was quantified using the BCA assay (Beyotime, China). Samples were incubated for 10 min at 100 °C in a reducing agent and loading buffer. Concentrations of primary antibodies used in the western blotting were as follows: anti-Flag antibody (Sigma, 1:4000), anti-PTEN antibody (Cell signaling Technology, 1:1000), anti-GFAP antibody (Abcam, 1:1000), anti-CD81 antibody (Abcam, 1:200), anti-CD63 antibody (Abcam, 1:1000) and anti-GAPDH antibody (Abcam, 1:1000).

Primary antibodies utilized in our study: anti-HSP70 antibody (1 : 1000, Proteintech, Chicago, USA), anti-CD63 antibody (1 : 1000, ABclonal, Boston, USA), anti-CD81 antibody (1 : 1000, Abcam, MA, USA); secondary antibodies include HRP-labeled goat antirabbit IgG (1 : 1000, Beyotime, Shanghai, China). A chemiluminescence kit was purchased from Millipore (Darmstadt, Germany).

Western blots were performed following SDS PAGE using standard protocols involving anti-CD81 antibody (Cat# ab232390) from Abcam, Waltham, MA, USA; anti-TSG101 antibody (Cat# SC-7964), anti-HSP70 antibody (Cat# SC-24) and anti-Calnexin antibody (Cat# SC-23954) from Santa Cruz Biotechnology, CA, USA. Following incubation with appropriate secondary antibody, proteins were detected by chemiluminescence using Clarity Max ECL substrate (Bio-Rad Laboratories, Hercules, California, USA) on a Chemidoc-MP Gel imaging system (Bio-Rad Laboratories, Hercules, California, USA).