Casp3

Proteins from myocardial and renal tissues were extracted with lysis buffer (Keygen, China) supplemented with phenylmethylsulfonyl fluoride, phosphatase inhibitors, and protease inhibitors. Equal amounts of the protein samples were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose filter membrane (GE Healthcare Life Sciences, UK). After blocking in 5% bovine serum for 2 h, the membranes were incubated at 4°C overnight with primary antibodies against caspase 3 (CASP3; 1:1,000, Proteintech, USA), albumin (ALB; 1:1,000, Affinity, USA), epidermal growth factor receptor (EGFR; 1:1,000, Affinity, USA), annexin A5 (ANXA5; 1:1,000, Proteintech, USA), peroxisome proliferator activated receptor γ (PPARG; 1:1,000, Proteintech, USA), and GAPDH (1:1,000, Proteintech, USA). Then the membranes were washed 3 times with Tween 20/Tris-buffered saline and incubated with the secondary antibody (LI-COR Biosciences, USA) for 2 h at room temperature. After washing 3 times with Tris-buffered saline, the fluorescence signal of the secondary antibody could be detected by the Odyssey fluorescence imaging system (LI-COR Biosciences, USA).

Cells were collected 48 h after transfection and cell lysates were prepared using ice-cold Tris buffer (20 mmol/l Tris; pH 7.5) containing 137 mmol/l NaCl, 2 mmol/l EDTA, 1% Triton X, 10% glycerol, 50 mmol/l NaF, 1 mmol/l DTT, PMSF, and a protein phosphatases inhibitor (Applygen Tech. Beijing, China). For extracellular signalregulated kinase (ERK) signaling analysis, cells were starved with serum-free medium for 24 h after transfection. These cells were then stimulated with medium containing 10% serum for 60 min before collection. To analyze the sensitivity of AZD0156, cells were exposed to UV 20 mJ/cm 2 for 2 h [Citation39] before treatment with 0.5 μM/l AZD0156 or ethanol (control), and cells were collected after 24 h treatment. western blot was performed as described previously [Citation34]. Quantification of blots was performed with Image J.
Primary antibodies were as follows: TMEM176A (Sigma-Aldrich), cleaved CASP3 (Protein Tech Group, IL, USA), Bcl2 (Protein Tech Group), CASP3 (Protein Tech Group), Bax (Protein Tech Group), MMP-2 (Protein Tech Group), MMP-9 (Protein Tech Group), cyclin B1 (Protein Tech Group), CDC2 (Protein Tech Group), ATM (HuaXingBoChuang, China),γ-H2AX (HuaXingBoChuang), p-CHK2 (Zhengneng, China), ERK1/2 (Protein Tech Group), p-ERK1/2 (Cell Signaling Technology, MA, USA), SAR1A (Protein Tech Group) and β-actin (Beyotime Biotech, Nanjing, China).