Lc3 1 2

Proteins (30 μg) derived from treated and untreated hDPSCs with and without 0.1 µL of rapamycin (Sigma Aldrih, Milan, Italy) were processed as previously described [42 (link)]. All antibodies used for the Western blot procedure were purchased from Santa Cruz Biotechnology. After protein separation, saturated sheets were incubated overnight at 4 °C with P62 (1:200, Santa Cruz Biotechnology), LC3-I/II (1:1000, Santa Cruz Biotechnology), ERK (1:750, Santa Cruz Biotechnology), pERK (1:750, Santa Cruz Biotechnology), Beclin1 (1:1000, Santa Cruz Biotechnology), and β-actin (1:1000, Santa Cruz Biotechnology).
Samples were then washed and incubated in secondary antibody diluted 1:1000 in 1× TBS, 5% milk, 0.05% Tween-20. Protein-specific bands were visualized via the electrochemiluminescence (ECL) method [43 (link)].

The hearts or EPCs were harvested and homogenized in lysis buffer for Western blot analysis. Total protein concentrations were determined using a BCA protein assay kit (Pierce Co, IL). Fifteen µg of protein were subjected to SDS-PAGE on 10% polyacrylamide gels and transferred to a nitrocellulose membrane. The blot was probed with Sirt3, VEGFR2, Akt and eNOS (1∶1000, Cell Signaling, MA), VEGF, CXCR-4, gp91^phox, p47^phox, LC3-I/II and beclin-1 (1∶1000, Santa Cruz, CA) antibodies. The membranes were then washed and incubated with a secondary antibody coupled to horseradish peroxidase and densitometric analysis was carried out using image acquisition and analysis software (TINA 2.0).

Antibodies against VDAC1, Cytc, Hsp60, Parkin, PINK1, p62, p70s6k, P-p70s6k, and LC3 I/II were obtained from Santa Cruz Biotechnology (Dallas, TX, US); antibody to β-actin and all secondary antibodies were obtained from Proteintech (Wuhan, Hubei, China). The specific assay procedures were as previously described^{27 (link)}.

Proteins of cells and tissues were isolated using a RIPA buffer containing protease and phosphatase inhibitor cocktail. Total protein was quantified using the BCA method (Fude Biological Technology, China, FD2001). Equal amounts of protein from each group were separated through an SDS-polyacrylamide gel. The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology. FTO (AF6936) and N6-Methyladenosine (m6A) Rabbit Polyclonal Antibody (AF7407) were from Beyotime. Protein half-life assays were performed according to our previous protocol (22 (link)). Briefly, cell lysates from the control or Cana group were collected at different time points after cycloheximide (CHX) administration and subjected to immunoblot analyses with the anti-STAT6 and anti-GAPDH antibodies. The prestained protein marker (Vazyme Biotech Co., Ltd., MP102-01) was used to identify the specific bands.