P38 mitogen activated protein kinase

Recombinant human thioredoxin-1 (TXN; E. coli-derived) was purchased from R&D Systems Inc. (Minneapolis, MN, USA). PE-conjugated anti-mouse Scal-1 and PerCP-5.5 lineage cocktail antibody was obtained from BD Biosciences (San Jose, CA, USA). FITC-conjugated anti-mouse CD48 and APC-conjugated anti-mouse CD 150 were obtained from Biolegend (San Diego, CA, USA). PE-Cyanine 7-conjugated anti-mouse CD117 (c-Kit) was purchased from eBioscience Inc. (San Diego, CA, USA). Senescence β-galactosidase (SA-gal) staining kit, γ-H2AX, p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK (Thr180/Tyr182), p16, β-actin, and the corresponding secondary antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

At the end of the experiment at 12 weeks of age, the heart was excised. Western blot analysis was carried out using homogenates from the heart tissues. Proteins were separated and transferred to membranes using standard protocols, after which they were probed with antibodies against prorenin and renin (1:100; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), (pro)renin receptors (1:100; Santa Cruz Biotechnology, Inc.), angiotensinogen (1:200; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA), angiotensin II AT1 receptor (1:200; Enzo Life Sciences, Inc., Farmingdale, NY, USA), extracellular signal-related kinases (ERK)1/2 (1:100; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated (p)-ERK1/2 (1:100; Cell Signaling Technology, Inc.), transforming growth factor (TGF)- β1 (1:200; Santa Cruz Biotechnology, Inc.), p38 mitogen-activated protein kinase (MAPK) (1:100; Cell Signaling Technology, Inc.), p-p38MAPK (1:100; Cell Signaling Technology, Inc.), heat shock protein (HSP)27 (1:100; Santa Cruz Biotechnology, Inc.) and p-HSP27 (1:100; Santa Cruz Biotechnology, Inc.). The blots were visualized by means of chemiluminescence (ECL; GE Healthcare UK Ltd., Amersham Place, Buckinghamshire, England), and the signals were quantified by densitometry. GAPDH (analyzed with an antibody from Cell Signaling Technology, Inc.) served as the loading control.

Asiatic acid (#A2612), bafilomycin A1 (BA1) (#B1793), compound C (#P5499), CQ (#C6628), ammonium chloride (NH₄Cl, #A9434), PPD (#P0031), rapamycin (Rap, #553210), and staurosporine (#S5921) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against the autophagy marker light chain 3 (LC3) A/B (#12741), p62 (#8025), mechanistic target of Rap (mTOR) (#2983), phosphorylated (p)-mTOR (#5536), Bcl-xL (#2764), Bcl-2 (#15071), p38 mitogen-activated protein kinase (MAPK, #8690), p-p38 MAPK (#4511), p-c-Jun N-terminal kinase (JNK, #4668), JNK (#9252), AMP-activate protein kinase (AMPK, #5831), and p-AMPK (#2535) were obtained from Cell Signaling Technology (Danvers, MA, USA). Beclin-1 (#SAB1306537) and tubulin-α (#SAB2102603) antibodies were purchased from Sigma-Aldrich. The lysosomal membrane-associated protein (LAMP)-2 (#NBP222217) antibody was purchased from Novus Biologicals (Centennial, CO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse (#31430) and anti-rabbit (#31460) antibodies were purchased from Invitrogen (Carlsbad, CA, USA).