Horseradish peroxidase labelled secondary antibodies

Antibodies specific for LC3B, p62, mTOR, Phospho‐mTOR, p70S6K, Akt, Phospho‐Akt, Cleaved caspase 3 (C‐caspase3), Bax, Cox‐2 and TNF‐α were purchased from Cell Signalling Technologies. Antibodies against iNOS, Bcl‐2, Iba1 and Arg‐1 were purchased from Abcam. The antibody of p‐p70S6K was obtained from Santa Cruz Biotechnology, and the antibody of CD16/32 was purchased from BD Biosciences Pharmingen. AS‐IV was purchased from MedChemExpress. Lipopolysaccharide (LPS) and tert‐butyl hydroperoxide (TBHP) were purchased from Sigma‐Aldrich. 3‐methyladenine (3‐MA) was purchased from Selleckchem. 4', 6‐diamidino‐2‐phenylindole (DAPI) was obtained from Beyotime. The polymerase chain reaction (PCR) primers were synthesized by Sangon Biotech, and other reagents used in the quantitative real‐time polymerase chain reaction (qPCR) were ordered from Takara Biomedical Technology. Horseradish peroxidase‐labelled secondary antibodies were purchased from Abcam, and AlexaFluor 594 and AlexaFluor 488 AffiniPure secondary antibodies were purchased from Yeasen.

Exosomes, cells or tissues were lysed, and their protein contents were measured using the Micro BCA Protein Assay Kit (Thermo, Waltham, MA, USA). The lysates were centrifuged, subjected to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked and incubated with the specific primary antibody overnight at 4°C and horseradish peroxidase‐labelled secondary antibodies (dilution 1:5000–10,000; Abcam, Cambridge, MA, USA) were added. Protein expression was visualized using enhanced chemiluminescence reagents (Amersham, Piscataway, NJ, USA) and the ChemiDoc XRS system (Bio‐Rad, Hercules, CA, USA). The primary antibodies used are as follows: CD9 (#ab92726, 1:2000; Abcam), CD63 (#ab134045, 1:2000; Abcam), Alix(#ab117600, 1:2000; Abcam), TSG101(#ab30871, 1:1000; Abcam), CD105(#ab107595, 1:500; Abcam), phosphatase and tensin homolog (PTEN; #9559, 1:1000; Cell Signaling Technology, CST, Beverly, MA, USA), phospho‐PTEN (#9549, 1:1000; CST), Akt (#2920, 1:2000; CST), phospho‐Akt (#4058, 1:1000; CST), Bad (#9268, 1:1000; CST), Bcl‐2 (#3498, 1:1000; CST), Bax (#5023, 1:1000; CST), caspase‐3 (#9665, 1:1000; CST) and cleaved caspase‐3 (#9664, 1:1000; CST). The antibody for GAPDH (#9485, 1:2500; Abcam) was used as a control.