Total proteins were extracted from treated cells and myocardial infarct tissue using a RIPA buffer with protease and phosphatase inhibitors and used for western blotting as previously described 15 (link). The proteins' concentrations were determined with a Bradford protein assay kit (Bio-Rad, Richmond, CA, USA) using BSA as a protein standard. Proteins were separated by SDS 10% PAGE, blotted on Hybond-C membranes (Amersham Biosciences, GE Healthcare, France) and incubated with various antibodies: PHD2 (Bioworld, catalog # BS6184), HIF-1α (Bioworld, catalog # BS3514), VEGF (Santa Cruz, catalog # sc-13083), bFGF (Abcam, catalog # ab8880), caspase-3 (Santa Cruz, catalog #sc-1225) and GAPDH as an internal control (Santa Cruz, catalog # sc25778). ECL HRP Linked Rabbit IgG (Santa Cruz, catalog # sc2793) was used as the secondary antibody. The density of the respective bands was quantitated using a densitometer with AlphaView Software for FluorChem Systems (ProteinSimpleTM).
Hif 1α
Manufactured by Bioworld Technology
Sourced in United States, China
HIF-1α is a protein that plays a central role in the cellular response to hypoxia, or low oxygen conditions. It functions as a transcription factor, regulating the expression of genes involved in various cellular processes, such as angiogenesis, metabolism, and cell survival.
Automatically generated - may contain errors
Lab products found in correlation
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Bioworld Technology (2 mentions)
γ h2ax, by Bioworld Technology (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Hybond c membrane, by Cytiva (1 mentions)
Basic fibroblast growth factor (bfgf), by Abcam (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
Hybond, by Cytiva (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Total caspase 3, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Myogenin, by R&D Systems (1 mentions)
Elongin c, by Santa Cruz Biotechnology (1 mentions)
Elongin b, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Ge imagequant 350, by GE Healthcare (1 mentions)
13 protocols using hif 1α
1
Protein Expression Analysis in Myocardial Infarction
2
Western Blot Analysis of Cell Signaling
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Western blotting was performed as described in a previous study 60 (link). Antibodies targeting the following proteins were used: CD63 and PDCD4 (Novus, CO, USA); BCL-2, total caspase3, cleaved- caspase3, p-AKT, AKT, PTEN, p-P65 and P65 (Cell Signaling Technology, MA, USA); HIF-1α (Bioworld, MN, USA); NGAL (R&D systems, USA); myogenin (R&D systems, USA) and GAPDH (Santa Cruz Biotechnology, TX, USA).
3
Isolation and Use of Wogonin in Research
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Wogonin was isolated from S. baicalensis Georgi according to the protocols reported previously with slight modifications [21 (link)]. Wogonin was dissolved in dimethylsulfoxide (DMSO) as a stock solution and stored at −20°C. Further dilution was performed in culture medium prior to each experiment (DMSO%<0.1%). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Fluka and dissolved in 0.01 M PBS. The enzyme-linked immunosorbent assay (ELISA) kit for VEGF was purchased from BosterBio (Pleasanton, USA). The rabbit polyclonal antibodies to c-Myc, HIF-1α, VEGF, VHL, CUL2, Flag-tag and PIASy were purchased from Bioworld. The mouse polyclonal antibody to β-actin was obtained from Boster. Other antibodies to Rbx1, Elongin C, Elongin B and HA were from Santa Cruz Biotechnology. IRDyeTM 800 conjugated secondary antibodies were obtained from Rockland Inc. Five-to-six-week-old female BALB/c-nude mice (SLACAS, Shanghai, China) were used for xenograft assays. The mice were fed ad libitum throughout the experimental period.
4
Cardiac Protein Extraction and Western Blot
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Total protein was extracted from cardiac muscle tissues using RIPA buffer supplemented with PMSF. The protein concentration was quantified using a BCA protein assay kit (DingguoChangsheng Biotech Co., Beijing, China). The protein was separated in 6% or 10% SDS-PAGE and transferred to the NC membrane (Millipore Corp., Bedford, Mass, United States) by electroblotting. Then, membranes were blocked in 5% skim milk for 1 h at room temperature and incubated at 4°C overnight with the primary antibodies of HIF-1α, ACE, ACE2, and GAPDH (Bioworld, Atlanta, GA, United States). After washing with Tris-buffered saline containing 0.5% Tween-20 (TBST), membranes were incubated with the secondary antibody for 2 h at room temperature. The blots were developed using the SuperSignal West Pico chemiluminescent substrate kit (Thermo Scientific, Rockford, IL, United States) and visualized by an ECL chemiluminescent system (GE ImageQuant 350, GE Healthcare).
5
Investigating Pancreatic Tumor Progression in KPC Mice
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KPC mice, LSL-KrasG12D mice, and Trp53fl/fl mice were purchased from the Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China. The feeding, breeding, and genotypic identification of the KPC (LSL-KrasG12D/+, Trp53fl/+, and Pdx1-Cre) mice were performed as previously described23 (link). To explore tumor formation kinetics, KPC mice were sacrificed at different time points (6, 10, 14, and 18 wk). To investigate the antitumor effects of RSV in vivo, 8-wk-old KPC mice were divided into two groups and treated daily with or without 50 mg/kg RSV and sacrificed at 16 wk based on our previous study24 (link). The methods used for tissue preparation and histopathologic analysis, i.e., hematoxylin and eosin (H&E) staining, immunohistochemical staining, and Masson’s trichrome staining, were performed in accordance with our previously reported methods25 (link). Briefly, standard immunohistochemistry was performed using HIF-1α (Bioworld, St Louis Park, MN, USA), α-SMA (Sigma), VEGF-A, IL-6, and SDF-1 (Abcam) antibodies. Masson’s trichome staining to assess fibrosis was carried out using a kit from Sigma according to the manufacturer’s protocol. The staining results were evaluated by two pathologists blinded to the clinical data, as described by Sinicrope et al.26 (link).
6
Purification and Characterization of Escin Derivatives
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SFAC (containing saponin constituents higher than 74.4%), escin Ia (C55H86O24, MW: 1131.26, purity ≥ 98%), escin Ib (C55H86O24, MW: 1131.26, purity ≥ 98%), escin IIa (C54H84O23, MW: 1101.23, purity ≥ 98%), escin IIb (C54H84O23, MW: 1101.23, purity ≥ 98%), escin IIIa (C55H86O23, MW: 1115.26, purity ≥ 98%) and escin IIIb (C55H86O23, MW: 1115.26, purity ≥ 98%) were obtained from Yinxing Pharmaceutical Technology Co., Ltd. (Nanjing, China) (Figure 1 ); β-aminopropionitrile (BAPN) was obtained from Sa'en Co., Ltd. (Shanghai, China); Transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) were obtained from R & D Systems (Minneapolis, USA); TRIzol and Lipofectamine 2000 reagents were obtained from Invitrogen (Carlsbad, USA); E-cadherin and LOXL2 antibodies were purchased from Genetex (Irvine, USA); HIF-1α and vimentin antibodies were purchased from BioWorld (Georgia, USA); α-SMA antibody was purchased from Abcam (Cambridge, UK); GAPDH monoclonal antibody was purchased from KangChen Bio-tech (Shanghai, China); HRP-conjugated secondary antibodies were purchased from Abbkine (Redlands, USA); Fetal bovine serum (FBS) was obtained from PAA (Linz, Germany). Other analytical reagent grade chemicals were obtained from Sinopharm Chemical Reagent Co. Ltd (Nanjing, China).
7
Immunohistochemical Analysis of Rat Hearts
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Formaldehyde-fixed rat hearts were dehydrated and then embedded in paraffin. Paraffin-embedded sections (5 μm) were dewaxed and incubated with the primary antibody HIF-1α (Bioworld, catalog # BS3514), VEGF (Santa Cruz, catalog # sc-13083) or CD31 (Santa Cruz, catalog # sc-1506). After visualization with diaminobenzidine (DAB), images were acquired under the inverted Olympus IX70 microscope (Olympus Inc. Melville, NY). The CD31 immunohistochemical staining was performed to determine the capillary density. The myocardial capillary density (MCD) was also calculated as previously described 19 (link).
8
Plasmid DNA Extraction and Cell Transfection
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Blood and Tissue DNA extraction kit was purchased from Tiangen; plasmid pEGFP-C1 was purchased from Dingguo biotechnology company; LA Tag enzyme, pMD19-T simple plasmid restriction enzyme ScaI and KpnI, DNA ligase,DNA marker, Reverse transcription kits were purchased from TaKaRa; Endotoxin-free plasmid extraction kit was purchased from QIAGEN; DMEM medium, fetal bovine serum were purchased from Gibco; Lipofectamine TM 2000 was purchased from Invitrogen;SsoFastTM EvaGreen Supermix was purchased from BIO-RAD, TFAP2c primary antibody, HIF-1α, anti-β-action antibody were purchased from Bioworld;EZ DNA Methylation-Gold Kit, methylation positive control and a negative control were purchased from ZYMO;HepG2 and H9C2 cell were conserved in our laboratory.
9
Western Blot Analysis of HIF-1α in Cultured Cells
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Total cellular protein from cultured cells was extracted with RIPA buffer (Beyotime Biotechnology, Shanghai, China), and the total protein concentration was determined using a BCA protein assay kit. NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher, Rockford, USA) were used as per the manufacturer’s instructions to extract nuclear and cytoplasmic proteins from the cells. Typically, 15–55 mg of total protein was separated using an 8% SDS-PAGE gel and transferred onto a PVDF membrane. The membranes were then blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies specific to HIF-1α (1:500, BioWorld Technology, USA), Lamin B1 (1:1000, Thermo Fisher, Rockford, USA), and β-actin (1:1000, Cell Signaling Technology, USA). After washing three times for 10 min each with 15 mL of TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1:2000) for 1.5 h at room temperature. The membranes were then washed again, three times for 10 min each with 15 mL of TBST. Finally, the protein bands were visualized using ECL reagent (Millipore, USA).
10
Sodium Hyaluronate-Tyramine Hydrogel Synthesis
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Sodium hyaluronate (HA) (>95%, Mw = 90 kDa), tyramine hydrochloride (Tyr·HCl), 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl), Hydrogen peroxide (H2O2, wt. 30%), N-hydroxysuccinimide (NHS), Horseradish peroxidase (HRP, 400–1,000 U/mg) and bovine testicular hyaluronidase were all purchased from MeiLun Co. Ltd. (Dalian, China). AL (dihydrochloride form; purity >99.9%) was obtained from Jiangsu Chia-tai Tianqing Pharmaceutical Co, Ltd (Nanjing, China). Crystal violet was purchased from Kelong Co. Ltd. (Chengdu, China). Polyclonal antibodies against Ki-67, VEGF-A, HIF-1α, and γ-H2AX were purchased from Bioworld Technology Co. Ltd. (Nanjing, China).
LLCs and human umbilical endothelial cells (HUVECs) were obtained from the experimental laboratory of Southwest Medical University (Luzhou, Sichuan, China) and cultured in a humidified 5% CO2 atmosphere at 37°C with Dulbecco’s Modified Eagle’s medium (HyClone; Thermo Scientific, Waltham, MA, USA), 10% fetal bovine serum (HyClone, Thermo Scientific), 0.1 mg/ml streptomycin and 100 U/ml penicillin.
LLCs and human umbilical endothelial cells (HUVECs) were obtained from the experimental laboratory of Southwest Medical University (Luzhou, Sichuan, China) and cultured in a humidified 5% CO2 atmosphere at 37°C with Dulbecco’s Modified Eagle’s medium (HyClone; Thermo Scientific, Waltham, MA, USA), 10% fetal bovine serum (HyClone, Thermo Scientific), 0.1 mg/ml streptomycin and 100 U/ml penicillin.
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