Caspase 7

Tianma Gouteng Decoction from Gastrodia elata 10 g, Uncaria 15 g (later fried), abalone shell 20 g (first fried), Gardenia 6 g, Scutellaria baicalensis Georgi 6 g, achyranthes root 12 g, Eucommia ulmoides Oliv. 12 g, Leonurus japonicus 12 g, Loranthus parasiticus 12 g, dried caulis of polygoni multiflori 12 g, and Poria cocos 12 g. All of the above ingredients were provided by the Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. TGD is concentrated into granules; each 1 g granule is equal to 10 g of raw materials and complies with the guidelines of Good Manufacturing Practices and Good Laboratory Practices formulated by Chinese government agencies. The TGD was dissolved in pure water to prepare solutions having a concentration of 200 mg/mL or 400 mg/mL for the experiment.
Saline was purchased from China Otsuka Pharmaceutical Co., Ltd. (Tianjin, China). The enzyme-linked immunosorbent assay (ELISA) kits, including prostacyclin (PGI2), thromboxane A2 (TXA2), angiotensin II (Ang II), and endothelin 1 (EDN1), were all obtained from Uscn Life Science, Inc. (Wuhan, China). The primary antibodies of GAPDH, AKT, p-AKT, caspase 8, caspase 7, caspase 3, and secondary antibodies were purchased from Proteintech Inc. (IL, USA). The antibodies of OPG, TRAIL, and DR5 were purchased from Abcam PLC (Cambridge, UK).

Proteins were separated by SDS-PAGE. After electrophoresis, gels were cut and transferred to PVDF membranes. Membranes were blocked with 5% w/V evaporated milk in PBST. Diluted primary antibodies were added and incubated overnight at 4 °C. Primary antibodies were against TRIM36 (1:1000, Sigma), BAX (1:10,000, Proteintech), and BCL2 (1:2000, Proteintech), caspase-3 (1:1000, Abclone), cleaved caspase-3 (1:200, Sigma), caspase-7 (1:5000, Proteintech), PARP1 (1:1000, Abclone), MMP-9 (1:500, Proteintech), cyclin D1 (1:1500, Proteintech), β-catenin (1:20,000, Proteintech), active β-catenin (1:1000, Cell signaling technology), c-JUN (1:2000, proteintech), Histone H3 (1:10,000, Proteintech), Actin (1:10,000, Proteintech). Thereafter, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) or HRP-goat anti-mouse IgG (H + L) secondary antibodies (Proteintech) for 50 min at room temperature. Chemiluminescence was measured using Super Signal West Atto (Thermo Fisher Scientific). Blots were imaged with Amersham Imager 600 software and analyzed using ImageJ software.