Anti lc3

TF, Beclin-1, and LC3B protein expression was analyzed by western blot analysis. Protein concentrations were estimated using the Lowry protein assay. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Following blocking with BSA, the membranes were incubated overnight at 4 °C with anti-rat TF, anti-Beclin1 and anti-LC3 (1:500, 1:500, 1:1000, respectively; Abcam (Shanghai) trading co., LTD, Shanghai, China). The membranes were then incubated with HRP-conjugated secondary antibody (originated from rabbits) for 2 h at room temperature and the targeted antigens were detected using enhanced chemiluminescence reagents. The targeted proteins were analyzed using the Lab-work image analysis software (Gene Company Limited, Hong Kong, China).

All reagents we used were commercially available. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen (Carlsbad, California). Recombinant human bFGF was purchased from Sigma (Sigma‐Aldrich, St. Louis, Missouri). Anti‐GFAP, anti‐bFGF, anti‐p62, anti‐NeuN, and anti‐GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Anti‐GAP43, anti‐LC3, anti‐Beclin‐1, and anti‐Nestin antibodies were purchased from Abcam (CB, United Kingdom). Goat anti‐rabbit and anti‐mouse IgG‐HRP, goat anti‐chicken IgY H&L, donkey anti‐goat IgG H&L were purchased from Santa Cruz Biotechnology. An enhanced chemiluminescence kit and CM‐DiI were purchased from Bio‐Rad (Hercules, California). Thapsigargin (TG) and 3‐methyladenine (3‐MA) were purchased from Sigma‐Aldrich. The autophagy activator rapamycin (RAPA) was purchased from Cell Signaling Technology. All other reagents were purchased from Beyotime Institute of Biotechnology (Shanghai, China) unless otherwise specified.

Exosomes or cells were lysed using lysis buffer (Beyotime Biotechnology, China) with freshly added phosphatase inhibitors and protease inhibitors (Roche, Penzberg, Germany). A BCA protein assay kit (ComWin Biotechnology, Beijing, China) was used to detect the protein concentration. Then, 20 μg denatured protein was added to an SDS-PAGE gel and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Munich, Germany). Then, 5% fat-free dry milk in Tris-buffered saline with Tween-20 (TBST) buffer was added to block the membranes for 1 h at room temperature. Afterward, the membranes were probed with the following primary antibodies overnight at 4°C: anti-TSG101, anti-CD9, anti-CD63 (Bimake, Houston, TX, United States), anti-LC3 (Abcam, Santa, United States), anti-Beclin1, anti-p-mTOR, anti-mTOR, anti-p-p70s6k, anti-p70s6k, anti-Rheb, and anti-GAPDH (Cell Signaling Technology, Danvers, MA, United States). After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature with gentle agitation. Proteins were detected using enhanced chemiluminescence (ECL) reagent (Bio-Rad, Munich, Germany) and scanned by a ChemiDoc MP gel imaging system (Bio-Rad, Munich, Germany). The mean pixel density of each strip was measured by Image Lab (Bio-Rad, Munich, Germany).

The cell samples were lysed by Radio-Immunoprecipitation Assay (RIPA) Buffer (Beyotime, China) containing phosphatase inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF) for protein extraction. The total cell proteins were denatured with SDS-PAGE loading buffer, separated by SDS-PAGE gel and transferred to Polyvinylidene Fluoride (PVDF) membrane (Millipore, USA). Then the membranes were blocked with 10% BSA and incubated with primary antibodies including anti-ULK1 (Abcam, USA), anti-Beclin-1 (Abcam, USA), anti-LC3 (Abcam, USA), p-Erk1/2 (CST, USA), Erk1/2 (CST, USA) and anti-β-actin (CST, USA) overnight at 4 °C. Subsequently, the blots were washed with TBST (Tris Buffered Saline with 0.05% Tween 20) three times and incubated with the Horseradish Peroxidase (HRP) conjugated secondary antibodies (1:10000, CST) for 45 min. The chemiluminescence reagent (Thermo, USA) was used to expose and identify the protein bands using ChemiDoc Touch System (Bio-Rad, USA), and quantitative analysis of the protein expression level was calculated relative to the β-actin control using the ImageJ software (NIH, USA).

Stable overexpression cells were grown to 90% confluence in 6-well plates and lysed in RIPA buffer supplemented with 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, and 1× protease inhibitor cocktail. The cell suspensions were incubated for 15 min on ice, followed by short sonication. The supernatants of the lysates were collected after centrifugation at 10,000 rpm for 10 min at 4 °C. The samples were heated with 1× SDS loading buffer at 98 °C for 5 to 10 min. Following 10% or 15% SDS–PAGE and wet blot analysis, the blots were probed with one of the following primary antibodies in TBST: anti-p-mTOR (phospho-S2448) (Bioworld, Nanjing, China, Cat. No. BS4706), anti-mTOR (S2442) (Bioworld, Cat. No. BS3611), anti-LC3 (Abcam, Cambridge, UK, Cat. No. ab51520), anti-p62 (Abcam, Cat. No. ab56416), anti-LAMP2 (Proteintech, Wuhan, China, Cat. No. 66301-1-Ig), or anti-GAPDH (Proteintech, Cat. No. 60004-1-Ig). The blots were incubated with horseradish peroxidase-conjugated secondary antibody after three washes, and the signals were visualized using a chemiluminescence (ECL) system (Engreen, Beijing, China, Cat. No. 29100).

Proteins were obtained from the hearts or cell extracts and were analysed using Western blot analysis, as previously described [33 (link)]. The following primary antibodies were used: anti-LC3 (1:500; Abcam, Cambridge, MA, UK), anti-p62 (1:500; Abcam), anti-Beclin1 (1:500; Abcam), anti-AMPK (1:1000; Cell Signaling, Danvers, MA, USA), anti-p-AMPK-Thr172 (1:1000; Cell Signaling), anti-mTOR (1:1000; Cell Signaling), anti-p-mTOR-Ser2448 (1:1000; Cell Signaling), ULK1 (1:1000; Cell Signaling), and p-ULK1-Ser757 (1:1000; Cell Signaling). Targeted bands were normalized to GAPDH to confirm equal protein loading. The Western blot bands were quantified using ImageJ 3.0 software.