Bcl2 associated x (bax)

Total protein was extracted from cells after transfection using lysis buffer for western and IP (KeyGen, KGP701, Nanjing, China), and protein concentration was measured using a BCA protein assay kit (Beyotime, P0010, Shanghai, China). Proteinsamples were electrophoresed using SDS-PAGE and then transferred for incubation at 4ºC overnight with the following specific primary antibodies: p53 (ImmunoWay, YT3528, TX, USA), Bax (ImmunoWay, YT0455, TX, USA), Bcl-2 (ImmunoWay, YM3041, TX, USA), active caspase-3 (Abcam, ab32042, Cambridge, UK), PTPN1 (Abcam, ab75856, Cambridge, UK), MAP3K11 (Abcam, ab51068, Cambridge, UK), JNK1/2/3 (Bimake, A5005, TX, USA), c-Jun (Bimake, A5730, TX, USA), and GAPDH (ImmunoWay, YM3445, TX, USA). The samples were then incubated with HRP-conjugated anti-rabbit IgG antibody (CST, #7074, MA, USA) at room temperature for 1 h. Finally, proteins were detected using ECL (KeyGEN, KGP902, Nanjing, China). GAPDH was used as an internal reference. Image J was used for quantification of all Western blot bands.

BCA protein assay kit (#23225) and Isopropylthio-β-D-thiogalactopyranoside (IPTG, #R0392) were purchased from Thermo Fisher Scientific (USA). β-actin (#ab 8226), HSP 70 (#ab 5439), Bid (#ab 10640), Cyt C (#ab 133504), VEGF (#ab 32152), and Caspase-3 (#ab 13847) antibodies were purchased from Abcam (USA). Bax (#YM 3619) and HIF-1α (#YT 2133) antibodies were purchased from ImmunoWay Biotechnology, Inc (USA). Calcein-AM and propidium iodide assay kit (#04511) and MTT (#M2128) were obtained from Sigma-Aldrich (USA).

All herbal medicines were provided by Guangyuan Hospital of Traditional Chinese Medicine (Guangyuan, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) were purchased from the Gibco Co. (Grand Island, NY, United States). BCA protein assay reagents, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) preparation kit, penicillin-streptomycin mixture, trypsin, phosphate buffer saline (PBS) and cell counting kit-8 (CCK-8) were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). Annexin V-FITC/PI assay kit was purchased from Multisciences (Lianke) Biotechnology Corporate Limited (Hangzhou, China). H₂O₂ was purchased from Chengdu Chron Chemicals Co. Ltd. (Chengdu, China). Primary antibodies for PI3K, phosphorylation-PI3K (p-PI3K), AKT, phosphorylation-AKT (p-AKT), Nrf2, Bcl-2, Bax and cleaved-caspase-3 (C- caspase-3) were obtained from the ImmunoWay Biotechnology Co. (Suzhou, China). The assay kits for MDA, SOD, CAT, and GSH-PX were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Ultrapure water purified by Millipore Ultra-pure Water Purifier (Millipore, Milford, MA, United States) was used. Other reagents were all of analytical grade.

Protein was extracted from hUC-MSC samples or liver tissue using radioimmunoprecipitation assay (RIPA) lysis buffer. The primary antibodies were insulin receptor substrate 1 (IRS-1), phosphoinositide 3-kinase (PI3K, 1:1200, Immunoway Biotechnology), protein kinase B (AKT, 1:1200, Immunoway Biotechnology), phosphoinositide-dependent protein kinase 1(PDK1, 1:1200, Immunoway Biotechnology), glucose transporter type 4 (GLUT4, 1:1200, Immunoway Biotechnology), B-cell lymphoma-2-associated X (BAX, 1:1200, Immunoway Biotechnology), proliferating cell nuclear antigen (PCNA, 1:1200, Immunoway Biotechnology), B-cell lymphoma-2 (BCL-2), cell cycle-associated proteins (Ki-67, cyclin A, cyclin E, 1:1200, Immunoway Biotechnology), A 160 kDa substrate of the Akt Ser/Thr kinase (AS160, 1:1200, Immunoway Biotechnology), cysteine protease protein (Caspase3, 1:1200, Immunoway Biotechnology) glyceraldehyde phosphate dehydrogenase (GAPDH, 1:3000), and β-actin (1:3000). The secondary antibodies conjugated to horseradish peroxidase (1:3000, Immunoway Biotechnology). The normalization of other proteins was performed using β-actin and GAPDH western blot assays [19 (link)].

Proteins from cell lysates or tissue lysates were separated by a 10% SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After being blocked in 5% nonfat milk, protein blots were probed with a primary antibody followed by incubation with a peroxidase-conjugated secondary antibody. The primary antibodies included HIF-1α (1:1000; ImmunoWay), Bax (1:1000; ImmunoWay), Bcl-2 (1:1000; ImmunoWay), caspase3 (1:1000; ImmunoWay), cleaved-caspase3 (1:1000; ImmunoWay), and PTEN (1:500; ImmunoWay). Chemiluminescence was detected by the ECL-plus kit (Beyotime). Band intensity was quantified by Image J software.

The ground ovarian tissues were lysed using RIPA buffer (Biotechwell, Shanghai, China). Total proteins were isolated by centrifuging the lysates at 12,000 rpm for 5 min. The protein concentrations were determined using a bicinchoninic acid kit (Biotechwell). The protein samples were separated on 8–12% SDS-PAGE gels and then transferred to a polyvinylidene fluoride membrane. After incubation with 5% BSA at room temperature for 2 h, the membrane was incubated with primary antibody against TLR (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (1 : 1000; Affinity, Scoresby, Victoria, Australia), collagen type I alpha 1 chain (Col1A1; 1 : 1000; Affinity), α-SMA (1 : 1500; Servicebio), Bax (1 : 1500; ImmunoWay Biotechnology, Plano, TX, USA), p-p65 (1 : 1000; ImmunoWay Biotechnology), MyD88 (1 : 1000; ImmunoWay Biotechnology), p65 (1 : 1000; Affinity), or GAPDH (1 : 2000; Biotechwell) overnight at 4°C. After incubation with HRP-conjugated secondary antibody (1 : 2000; Jackson Immuno, West Grove, PA, USA) at room temperature for 2 h, the protein bands were detected using an enhanced chemiluminescence kit (Biotechwell).