Anti p mtor

Different groups of GH3 and AtT20 cells were lysed on ice with RIPA buffer. The isolated exosomes can be directly mixed with the loading buffer. Then, protein lysates were electrophoresed on 10% SDS polyacrylamide gels, transferred onto PVDF membranes (Millipore, Burlington, MA, USA), and blocked with NcmBlot blocking buffer (NCM Biotech, Suzhou, China). Afterward, primary antibodies: anti-ONECUT2 (Biorbyt, Cambridge, UK, cat: orb49419, 1:1000), anti-PI3K (Proteintech, Wuhan, China, cat: 60225-1-Ig, 1:1000), anti-P-PI3K (Thermofisher, Waltham, MA, USA, cat: PA5-104853, 1:1000), anti-PKCδ (Proteintech, Wuhan, China, cat: 14188-1-AP, 1:1000), anti-P-PKCδ (Proteintech, Wuhan, China, cat: 29562-1-AP, 1:1000), anti-mTOR (Protentech, Wuhan, China, cat: 66888-1-Ig, 1:1000), anti-P-mTOR (Proteintech, cat:67778-1-Ig, 1:1000), anti-AKT (Proteintech, cat: 60203-2-Ig, 1:1000), anti-P-AKT (Proteintech, Wuhan, China, Cat: 80455-1-RR, 1:1000), anti-ERK (Proteintech, Wuhan, China, cat: 11257-1-AP, 1:1000), anti-P-ERK (Proteintech, Wuhan, China, cat: 80031-1-RR, 1:1000), anti-GAPDH (Proteintech, Wuhan, China, Cat: 60004-1-Ig, 1:1000), anti-CD63 (Santa Cruz, CA, USA, cat: sc-5275;1:200), anti-CD9 (Santa Cruz, CA, USA, cat: sc-13118; 1:200) were added at 4 °C overnight. Subsequently, secondary antibodies (1:5000; Proteintech, Wuhan, China) were incubated for 2 h.

Metformin was purchased from MedChemExpress (Monmouth Junction, NJ, USA). 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used: anti-P53, anti-P21, anti-PI3K, anti-p-PI3K, anti-AKT, anti-p-AKT, anti-4EBP1, anti-p-4EBP1, anti-Bcl−2, anti-BAX, anti-cleaved caspase−3, anti-HIF−1α, anti-BAK (Cell Signaling Technology, Danvers, MA, USA); anti-P16, anti-GAPDH, anti-mTOR, anti-p-mTOR (Proteintech group, Inc., Rosemont, IL, USA); anti-Drp1, anti-Mcl−1, anti-PINK1, anti-Parkin, anti-MMP1 (Abcam, Cambridge, MA, USA); anti-HO−1, anti-Nrf2(Sangon Biotech, Shanghai, China); the antibodies were diluted according to the manufacturer’s instructions.