P mtor ser2448

Western blotting (WB) was performed as described [25 (link)]. Antibodies were used as per the manufacturer’s instructions and were as follows: CEBPD (Abcam, ab245214; or Invitrogen, PA5-30262); HIF1α (Abcam, #ab179483): EGFR (Cell Signaling Technology, #4267); p-EGFR (Tyr1173) (Cell Signaling Technology, #4407), ERK1/2 (Cell Signaling Technology, #4695), p-ERK1/2 (thr202/tyr204) (Cell Signaling Technology, #4370), AKT (Cell Signaling Technology, #4685), p-AKT (Ser473) (Cell Signaling Technology, #4060), mTOR (Wanleibio, Shenyang, China, WL02477), p-mTOR (Ser2448) (Wanleibio, WL03694), STAT3 (Cell Signaling Technology, #9139), p-STAT3 (Ser 727) (Cell Signaling Technology, #9134), FN1 (Proteintech, 15613-1-AP), COL1A1 (Wanleibio, WL0088), TNC (Proteintech, 67710-1-Ig), β-actin (Wanleibio, WL01372). All WBs were performed in triplicate, and quantification of protein expression levels were performed by using the ImageJ software (National Institutes of Health, Bethesda, MD).

The supernatant was obtained from the MSC medium with or without GW4869 that were cultured for 3 days. The MSC medium with or without GW4869 that were cultured for 3 days was ultracentrifuged to get exosomes. The exosomes were collected and resuspended in sterile 1×PBS for future use.
LY294002 (20 μM), LY303511 (10 μM), Triciribine (10 μM), mTOR, HSP70, CD9, CD63, and ULK1 were purchased from Beyotime (Shanghai, China). 3-MA (10 mM) was purchased from Selleckchem (Houston, USA). GW4869 (10 μM) was purchased from Targetmol (Boston, USA). Chloroquine (20 μM) was purchased from Merck (New Jersey, USA). Dorsomorphin (10 μM) was purchased from Glpbio (California, USA). p-AMPK/AMPK, p-mTOR(ser2448) were purchased from Wanleibio (Shenyang, China). p-AKT/AKT1/2/3 were purchased from Abmart (Shanghai, China). Bax, Bcl-2, Caspase3, Lamp1, LC3B, Beclin1, p-S6 (ser235/236), and p62 were purchased from Cell Signaling Technology (Boston, USA). Ataxin-3 was purchased from Millipore (Massachusetts, USA). GAPDH and p-TFEB (ser142) was purchased from Bioss (Beijing, China). TFEB was purchased from Abcam (Massachusetts, USA). Detailed information is listed in the Supplementary Table.