Sodium new houttuyfonate (SNH) was purchased from Xi'an Kailai Biological Engineering Co., Ltd. (Xi'an, China). We used high performance liquid chromatography to quantitatively analyze SNH with a purity of 98% (Fig. S1† ). HepG2 cells were obtained from ATCC. Dulbecco's Modified Eagle Medium (DMEM) was purchased from Gibco (USA). Fetal bovine serum (FBS), MTT, penicillin and streptomycin all were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Annexin V-FITC/PI apoptosis detection kit, Apoptosis-Hoechst staining kit and DNA ladder extraction kit were from Shanghai Beyotime Biological Technology Co. Ltd. (Shanghai China). The primer of GADPH, MMP9, VEGF and Bcl-2 were synthesized by Shanghai Sangon Biological Engineering Technology and Service Company. One step RT-PCR kit was bought from Qiagen (Germany). All other reagents were of the highest grade commercially available.
Bcl 2
Manufactured by Sangon
Sourced in China
Bcl-2 is a laboratory equipment product. It is a protein that regulates apoptosis, or programmed cell death, within cells. Bcl-2 acts as an anti-apoptotic regulator, helping to maintain cell survival.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 associated x (bax), by Sangon (4 mentions)
Cyclin e, by Sangon (2 mentions)
Wortmannin wort, by Merck Group (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Gadph, by Sangon (1 mentions)
Mmp 9, by Sangon (1 mentions)
Vascular endothelial growth factor (vegf), by Sangon (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
Cyclind1, by Sangon (1 mentions)
Odyssey two color infrared laser imaging system, by LI COR (1 mentions)
Inverted phase contrast microscope, by Nikon (1 mentions)
Nu nu nude mice, by Charles River Laboratories (1 mentions)
Bufalin, by Yuanye Bio-Technology (1 mentions)
β actin, by Sangon (1 mentions)
Rapamycin, by Beyotime (1 mentions)
Caspase 3, by Sangon (1 mentions)
9 protocols using bcl 2
1
Quantitative Analysis of Sodium New Houttuyfonate in HepG2 Cells
2
Lung Cancer Protein Expression Analysis
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Whole cell extractions were generated using M‐PER lysis buffer (Pierce, Rockford, IL, USA) from lung cancer cells, and protein concentrations were determined by BCA protein assay kit (Pierce). Standard Western blotting was performed with the routine approach. Primary WDR79 antibody (Bethyl Laboratories, Inc.) was diluted at 1:2000 in 5% blocking milk (Bio‐Rad, Hercules, CA, USA). Bax, Bcl‐2, Cyclin D1, cyclin E, pRb, PRAP, Bcl‐2, CYCs (Sangon Biotechnology, Shanghai, China) antibodies were diluted at 1:500. Caspase3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted at 1:1000. GAPDH 1:10,000 (KangChen Bio‐tech Inc., Shanghai, China) was used as an internal control.
3
Bufalin and Rapamycin Inhibit Esophageal Cancer
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The ESCC cell line, ECA109, was provided by the Scientific Research Center of the Fourth Hospital of Hebei Medical University (Hebei, China). A total of 36 Nu/Nu nude mice (half male and half female, aged 4–6 weeks, weighed 18–22 g) purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Our study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University. Bufalin was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China) and rapamycin was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). mTOR, p70S6K, 4EBP1, cIAP1, caspase-3, Bad, Bcl-2, β-actin upstream and downstream primers were all from Shanghai Sangon Biological Engineer ing Co., Ltd., (Shanghai, China). Rabbit anti-mTOR (1612-1), p70S6K (T2921), p-p70S6K (ab2571), 4EBP1 (1557-1), p-4EBP1 (2250-1), cIAP1 (3302-1), Bad (1541-1), Bcl-2 (1017-1) and β-actin monoclonal antibodies, and rat anti-active caspase-3 monoclonal antibody (1476-1) were obtained from Epitomics (Burlingame, CA, USA). The Alpha Innotech UV gel imaging system was from Alpha Innotech Co., (San Leandro, CA, USA) and the Odyssey two-color infrared laser imaging system was from LI-COR Biosciences (Lincoln, NE, USA) The inverted phase contrast microscope was from Nikon (Tokyo, Japan).
4
Ivermectin Cytotoxicity and Apoptosis Assay
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Ivermectin (95.9% purity) was purchased from (Cato Research Chemicals Inc., Eugene, OR, USA) 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT), Rhodamine123 (Rh‐123), 4′6‐diamidino‐2‐phenylindole (DAPI), N,N,N′,N′‐tetramethylethylenediamine, dimethylsulfoxide (DMSO), phenylmethanesulfonyl fluoride (PMSF), 2′,7′‐Dichlorodihydrofluorescein diacetate (DCFH‐DA), propidium iodide (PI), RIPA lysis buffer were purchased from Sigma (Beverly, MA, USA). IVM is dissolved in DMSO and diluted to the appropriate concentration in using cell culture medium. It should be noted that the final concentration of DMSO in the cell culture medium should be less than one thousandth. Antibodies such as cytochrome c, caspase‐9, caspase‐3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Bax, Bcl‐2, PARP, p53, CDK4 and CyclinE were brought from Sangon (Shanghai, China). Secondary anti‐rabbit antibody was from Sangon Biotech Co. Ltd. Other common chemicals and reagents were purchased from Shanghai Titan‐chem Co. Ltd. (Shanghai, China).
5
Evaluating Apoptosis Pathway in HT-29 Cells
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HT-29 cells (5 × 10 5 cells/mL) were seeded in 6-well plates and treated with 5 mg/mL IOP60b for 0 h, 24 h, 48 h, 72 h, respectively. Total RNA was extracted using total RNA extraction kit (GeneMark ™ ) and then reversed to cDNA by using the All-in-One ™ First-Strand cDNA Synthesis Kit (GeneCopoeia, USA) according to the manufacturer's protocol. Real-time PCR was performed with 2 × Es Taq MasterMix (Dye) (CWBIO, Beijing, China). The following primers (Sangon Biotech, Shanghai, China) were used for amplification: Bcl-2 forward 5′-CAG CTG CAC CTG ACG CCC TT-3′ and reverse 5′-GCC TCC GTT ATC CTG GAT CC-3′; Bax forward 5′-TTC TGA CGG CAA CTT CAA CTG-3′ and reverse 5′-TGA GGA GGC TTG AGG AGT CT-3′; Caspase-3 forward 5′-ACA CAG TAT GGC GGC AGA G-3′ and reverse 5′-AGA CAG GCA ACA GAG CAC AT-3′; βactin forward 5′-CCT CTA TGC CAA CAC AGT GC-3′ and reverse 5′-ATA CTC CTG CTT GCT GAT CC-3′.
6
Western Blot Analysis of Apoptosis Proteins
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Cells were lysed for 10 min with ice-cold lysis kit (Beyotime Institute of Biotechnology, China) supplemented with a protease inhibitor mixture (Sigma). An 8-min centrifugation was performed at 12,000 × g to collect the supernatant whose protein concentration was measured using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). A total of 30 µg of proteins from each sample were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, USA). The membranes were then blocked with 5% (w/v) non-fat milk (CWbio, China) in PBST for 2 h at RT. Next, the blots were incubated with rabbit polyclonal antibodies against human STC (1 : 500 in PBST; Abcam), Bcl-2 (1 : 600; Sangon Biotech), Bax (1 : 600; Sangon Biotech), caspase-3 (1 : 600; ABclonal Biotechnology, China), and GAPDH (1 : 5,000; Abcam) at RT for 2 h. The membranes were subsequently washed six times with PBST for 5 min at RT and then incubated with goat anti-rabbit IgG-HRP for 2 h at RT (1 : 8,000; CWbio). The immunoreactive bands were visualized using an Enhanced chemiluminescence (ECL) kit (CWbio).
7
Myocardial Protection Against Ischemia-Reperfusion Injury
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Streptozotocin (STZ) and wortmannin (Wort) were purchased from Sigma-Aldrich (St. Louis, MO, USA); dexmedetomidine (DEX) was purchased from Hengrui Pharmaceutical Co. (Jiangsu, China). Creatine kinase isoenzymes (CK-MB), lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) assay kits were obtained from Jiancheng Institute of Biotechnology (Nanjing, China). Primary antibodies obtained from Cell Signaling Technology Inc. (Danvers, MA, USA) were as follows: GSK-3β (BF0695), p-GSK-3β (AF2016), Akt (9272S), p-Akt (4060S), and β-actin (8H10D10). Rabbit anti-mouse and goat anti-mouse secondary antibodies were obtained from Boston Co. Ltd. (Wuhan, China). Bcl-2, Bax, and β-actin primers were obtained from Sangon Biotech Co. Ltd. (Shanghai, China) (Table 1 ).
8
Western Blot Analysis of Apoptosis Markers
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Protein concentrations were measured using BCA kit (Beyotime). Equal amounts of proteins were subjected to 10-15% SDS-PAGE and transferred to NC membrane. Antibodies against ST6Gal-I (Abcam, 1:500), p38, pp38, caspase-3 or GAPDH (Bioworld, 1:300, 1:300, 1:400, 1:10,000), Bcl-2, Bax, Bad or PARP (Sangon, 1:500), ctochrome c or caspase-9 (Proteintech, 1:500) were used as the primary antibodies. The detection was performed using ECL kit. Densitometry of proteins was analyzed with Gel-Pro software.
9
Cardioprotective Effects of DEX
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DEX was purchased from Jiangsu Nhwa Pharmaceutical Corporation Ltd. (Jiangsu, China). Wortmannin (Wort) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Rat CK-MB isoenzyme enzyme linked-immunosorbent assay (ELISA) kit (cat. no. E006), LDH assay kit cat. no. A020-2), cTnI ELISA kit (cat. no. H149-2), SOD assay kit (cat. no. A001-3) and MDA assay kit (cat. no. A003-1) were obtained from Jiancheng Institute of Biotechnology (Nanjing, China). TRIzol® reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Bcl-2, Bax and β-actin primers were acquired from Sangon Biotech Co., Ltd. (Shanghai, China). The following primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers MA, USA): Mouse anti-GSK-3β (1:2,000 dilution; cat. no. BF0695), rabbit polyclonal anti-p-GSK-3β (1:2,000 dilution; cat. no. AF2016), rabbit polyclonal anti-Akt (1:1,000 dilution; cat. no. 9272S), rabbit polyclonal anti-p-Akt (1:1,000 dilution; cat. no. 4060S) and rabbit polyclonal anti-cleaved caspase-3 (1:2,000 dilution; cat. no. 9661S). Horseradish peroxidase (HRP)-linked anti-mouse immunoglobulin (IgG) (1:10,000 dilution; cat. no. BA1050), HRP-linked anti-rabbit IgG (1:10,000 dilution; cat. no. BA1054) and anti-β-actin (1:1,000 dilution; cat. no. BM0627) were acquired from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China).
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