C1053 100

Rat heart tissues and cell lysates were prepared using RIPA lysis buffer containing protease inhibitor (c1053-100, Applygen, China), and phosphatase inhibitors (p1260-1, Applygen, China). An equal amount of protein was separated on 10%–15% SDS-polyacrylamide gels and transferred onto PVDF membranes. After blocking with 5% nonfat milk at room temperature for 2 h, the PVDF membranes were incubated with the appropriate primary antibody, followed by a secondary antibody. The PVDF membrane was exposed to ECL reagents (p1020-25, Applygen, China) to visualize the protein signals. The antibodies used for Western blot were as follows: anti-PI3K (1F6A7, Proteintech, USA), anti-Akt (2C5D1, Proteintech, USA), anti-Phospho–PI3K (CY6427, Abways, China), anti-Phospho–Akt (P31749, Abways, China), anti-VEGFA (ab 214424, Abcam, USA), anti-ARG-1 (P05089, Abways, China), anti-mTORC1 (Q6UUV9, Abways, China), anti-CD206 (JF0953, HuaAn, China), anti-rabbit IgG H and L (ab0101, Abways, China), and anti-mouse IgG H and L (ab0102, Abways, China).

Western blot analysis was used to detect protein expression levels in heart tissues or cells. Briefly, heart tissues or cells were lysed in pre-cold RIPA buffer (c1053-100, APPLYGEN, China) with a 0.5% protease inhibitor (p1625, APPLYGEN, China) and 1% phosphatase inhibitor (p1260-1, APPLYGEN, China). The protein concentration of each sample was measured using a BCA kit (P1511-1, APPLYGEN, China). Equal quantities of the proteins from each group were loaded on 10% PAGE gel fast preparation kit (PG112, Epizyme, China), electrophoresed, and transferred onto PVDF membranes (p2120-6, APPLYGEN, China). The membranes were incubated with primary and secondary antibodies and then treated with ECL (SQ201, Epizyme, China). The following antibodies were used: anti-TLR4 (19811-1-AP, Proteintech, United States), anti-MyD88 (D80F5, Cell Signaling Technology, Germany), Phospho-IκBα (Ser32) (14D4, Cell Signaling Technology, Germany), anti-IKBα (ab32518, Abcam, United States), Phospho-NF-κB p65 (Ser S536) (93H1, Cell Signaling Technology, Germany), anti-NF-κB p65 (D14E12, Cell Signaling Technology, Germany), and anti-GAPDH (ab8246, Abcam, United States), anti-mouse IgG H&L (AB0102; Abways, China) and anti-rabbit IgG H&L (AB0141; Abways, China). Finally, Image Lab software was used for band gray analysis.