Radio immunoprecipitation assay lysis buffer

Protein was extracted from an exosome using a Total Exosome Protein Isolation Kit (ThermoFisher). The cultured cells were lysed in Radio Immunoprecipitation Assay Lysis Buffer (Biosharp). The protein sample was separated using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE, Bio‐Rad) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The following antibodies were used as primary antibodies: Alix (ab186429, Abcam), TSG101 (A1692; Abclonal), CD63 (A19023, Abclonal), HSP70 (A12948; Abclonal), phospho‐PI3K (AP0854, Abclonal), PI3K (A11526, Abclonal), phospho‐Akt (AP0637, Abclonal), Akt (A11016, Abclonal), phospho‐FoxO1 (AP0172, Abclonal), FoxO1 (A2934, Abclonal), phospho‐FoxO3a (AP0684, Abclonal), FoxO3a (A0102, Abclonal), and β‐actin (AC026, Abclonal). Secondary antibodies were horseradish peroxidase (HRP) goat anti‐rabbit IgG (AS014, Abclonal) and goat anti‐mouse antibodies IgG (AS003, Abclonal). Visualization and analysis were performed using the iBright1500 system (Thermo).

Protein samples were extracted employing radioimmunoprecipitation assay lysis buffer (Biosharp) containing the protease inhibitor cocktail (Sigma–Aldrich) as directed by the manufacturer. After protein concentration was measured, proteins were first separated on a polyacrylamide gel and then deposited into polyvinylidene difluoride (PVDF) blots (Millipore, Billerica). Following the transfer, the blotting membranes were incubated with primary antibodies targeting NOD1 (Bioss Antibodies); phosphorylated P38, P38, phosphorylated ERK, ERK, phosphorylated JNK, JNK, Collagen I, Osterix, ALP, and Runx2 (ZEN BIO) were used in this study. Next, the membranes were treated with secondary antibodies for 2 h at room temperature. Then, protein bands were identified by enhanced chemiluminescence (ECL) reagents (Santa Cruz) treatment and exposure of x‐ray film. Protein bands were quantified using the Image J software.