Phospho akt

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).

Vaccarin was purchased from Shanghai Shifeng Technology (Shanghai, China). Streptozocin (STZ) was acquired from Sigma (St. Louis, MO, USA). FOXP2, AGGF1, total Akt, phospho-Akt, total Erk1/2, phospho-Erk1/2 antibodies were bought from Abclonal (Wuhan, Hubei, China). Total PI3K, β-actin antibodies were purchased from Abcam (Cambridge, MA, USA). The specific primers, control siRNA, and targeted FOXP2 siRNA were synthesized by Shanghai Sangon Biotech Co. Ltd. (Shanghai, China).

Extraction of proteins from primary cardiomyocytes and heart tissues was performed with RIPA lysis buffer (Beyotime, Shanghai, China) mixed with protease and phosphatase inhibitors (Beyotime, Shanghai, China). The protein was separated using 7.5–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. TBST was used to prepare 5% nonfat dry milk for 1 h at room temperature, and then it was incubated overnight with a primary antibody at 4 °C. The antibodies we used were as follows: RRM2 (ABclonal, A5255), Bcl-2 (Proteintech, 12789-1-AP), Phospho-mTOR (CST, 5536T), cleaved-Caspase3 (CST, 9661S), BECLin 1 (ABclonal, A7353), mTOR (CST, 2983T), AKT (Proteintech, 10176-2-AP), Phospho-Akt (Abclonal, AP1259), LC3B (Abclonal, A19665), GAPDH (Abcam, ab181602). Finally, HRP-conjugated secondary antibodies were used at room temperature for 1 h. Signals were visualized by enhanced chemiluminescence ECL (Thermo Scientific, Boston, MA, USA).