RNA extraction, cDNA preparation, and RT-qPCR were all performed utilizing the QIAGEN (Germantown, MD, USA) automated liquid handling workflow system (QIAcube HT and QIAgility). RNA from distal colonic tissue was isolated utilizing the QIAzol/chloroform extraction methodology. RNA was purified using the lithium chloride method as previously published [35 (link)]. Approximately 500 ng of extracted RNA was used to synthesize cDNA (QIAGEN’s RT2 First Strand Kit). RT-qPCR was performed utilizing QIAGEN’s Rotor-Gene Q thermo-cycler. Calculation of gene expression was conducted by comparing the relative change in cycle threshold value (ΔCt). Fold change in expression was calculated using the 2-ΔΔCt equation as previously described [36 (link)]. The following Taqman primers were used and obtained from Thermo Fisher Scientific: TNF-α (Mm00443258_m1), IL-1β (Mm00434228_m1), PAI-1 (Mm00435858_m1), MCP-1 (Mm00441242_m1), and CD40 (Mm00441891_m1). 18s rRNA from Thermo Fisher Scientific was used as a housekeeping gene for normalization of transcript expression (catalog no. 4319413E).
Pai 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
PAI-1 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is used to measure the levels of Plasminogen Activator Inhibitor-1 (PAI-1), a protein involved in the regulation of the fibrinolytic system. The core function of PAI-1 is to quantify the amount of this protein in a given sample.
Automatically generated - may contain errors
Lab products found in correlation
Adiponectin, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
α2 anti plasmin, by Cusabio (2 mentions)
18s rrna, by Thermo Fisher Scientific (1 mentions)
Tnfα mm00443258 m1, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Rotor gene q thermocycler, by Qiagen (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Mouse xl cytokine array, by R&D Systems (1 mentions)
Fusion sl2 3500 wl, by Vilber (1 mentions)
96 well plate reader, by Tecan (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Pai 1, by Santa Cruz Biotechnology (1 mentions)
Icam 1, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Leptin, by Merck Group (1 mentions)
Resistin, by Thermo Fisher Scientific (1 mentions)
16 protocols using pai 1
1
Gene Expression Analysis in Colonic Tissue
2
Cytokine Profiling in Peritoneal Fluid
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Peritoneal fluid was analysed with a Mouse XL Cytokine Array (R&D Systems) according to the manufacturer’s instructions. Chemiluminescence was detected with Fusion SL2-3500.WL (Vilber Lourmat, Suebia, Germany). Densiometric analysis of dots representing each cytokine was performed with ImageJ. Background signal was subtracted, and expression of each cytokine was normalized to its own positive control. Levels of IL-6 (R&D Systems), PAI-1 (Thermo Fisher Scientific), active tPA (LifeSpan BioScience, Seattle, USA) and were quantified by ELISA. Absorbance was measured at 450 and 540 nm using a 96-well plate reader (Tecan, Crailsheim, Germany).
3
Western Blot Protein Expression Analysis
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Protein expression levels in the HAECs were analyzed by western blotting as previously described (Wang et al., 2010 (link)). Primary antibodies against HO-1 (1:1,000) (Abcam, Cambridge, UK), ICAM-1 (1:500) (Cell Signaling Technology, Danvers, MA, USA), PAI-1 (1:1,000) (Santa Cruz Biotechnology), bFGF (1:500) (Millipore, Billerica, MA, USA), and GAPDH (1:5,000) (Santa Cruz Biotechnology) were used. Immunostaining was visualized using SuperSignal West Pico Chemiluminescent Substrate for HO-1 and PAI-1 and SuperSignal West Femto Maximum Sensitivity Substrate for ICAM-1 and bFGF (Thermo Scientific).
4
Multiplex Biomarker Profiling in Plasma
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Commercial ELISA kits were used to measure the following biomarkers in fasting plasma: adiponectin (#BMS2032), resistin (#BMS2040) and plasminogen activator inhibitor-1 (PAI-1) (#BMS2033) (Thermo Fisher Scientific, Waltham, MA, USA), leptin (# EZHL-80SK, EMD Millipore, Burlington, MA, USA) and visfatin (#EIA-VIS, RayBiotech, GA, USA). High sensitivity CRP was measured in fasting serum by immunoturbidimetry. IL-6 and TNF-α were measured in fasting plasma using the multiplex ELISA V-Plex Pro-inflammatory Panel I according to the manufacturer’s instructions (MesoScale Discovery, Rockville, MD, USA).
5
RT-qPCR Analysis of Adipose Gene Expression
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Total RNA was extracted from mWAT, adipocytes, or SVF using TRIsure reagent (Bioline Reagents Ltd., London, UK). cDNA was synthesized by the High Capacity cDNA Reverse Transcription Kit (Thermo Scientific) using 500 ng of total RNA. RT-qPCR was performed using StepOne Plus Real-Time PCR system with Taqman PCR master mix (Thermo Scientific). The primers used in this study were: Spred2 (Mn01223872_g1), tnfa (Mm00443258_m1), mcp-1 (Mm00441242_m1), adiponectin (Mm00456425_m1), leptin (Mm00434759_m1), pai1 (Mm00435858_m1), and rplp0 (Mm00725448_s1) (Thermo Scientific). The expression level of the gene of interest was normalized against rplp0 and expressed as fold-increases relative to the negative control for each treatment at each time point as previously described (17 (link), 33 (link)).
6
Antibodies Used in Extracellular Matrix Regulation
7
Quantifying Serum Biomarkers by ELISA
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Commercially available ELISA kits were performed in 96-well clear plates to quantify serum levels of transforming growth factor β (TGF-β, Invitrogen, Thermo Fisher Scientific, USA), plasminogen activator inhibitor type 1 (PAI-1, PeproTech, USA), FGFb (PeproTech, USA), and soluble urokinase-type plasminogen activator receptor (suPAR, RayBiotech, USA) according to manufacturer's instructions. The reported minimum detectable doses of TGF-β, PAI-1, FGFb, and suPAR were 8 pg/mL, 23 pg/mL, 63 pg/mL, and 15 pg/mL, respectively. Absorbance values were obtained after plate reading in spectrophotometer (Spectramax M3 Multi-Mode Microplate Reader®, Molecular Devices, USA) and analyzed using SoftMax Pro 6.0® (Molecular Devices, USA). All samples were tested in duplicate.
8
Quantifying Endothelial Cell Signaling
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HMEC-1 (1×105 cells) were transfected with indicated miRNA mimic or negative controls (NC mimic or inhibitors; 90 pmol) as described above and incubated in complete media at 37°C for 24 h. Cells were washed with SFM and incubated for 3 h in SFM (2 ml) treatments were begun by replacement with fresh SFM (1 ml) and additions, as indicated. Cells were treated with either PlGF (250 ng/ml) or fenofibrate (0.1 mM) overnight. The culture supernatant was collected and an aliquot (0.1 ml) was assayed for ET-1 (R&D Systems) and PAI-1 (Peprotech) release using an ELISA kit. The cells were collected by scraping and the pellet was assayed for protein content utilizing the Bradford method.
9
Saracatinib and PAI-1 Antagonist Protocol
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Saracatinib was purchased from MedChem Express (Shanghai, China). Human recombinant PAI‐1 and CCL5 (PeproTech, Rocky Hill, NJ, USA) were dissolved in culture medium, and a bioavailable PAI‐1 antagonist, PAI‐039 (PeproTech), was dissolved in DMSO (Sigma‐Aldrich) and stored at −80°C as a 1 mg/mL stock solution. The antibodies used included the following: BCRP/ABCG2 (Abcam, ab108312), PAI‐1 (Abcam, ab187262), CCL5 (Abcam, ab9666) and Ki‐67 (Abcam, ab15580).
10
Arsenic Trioxide Modulates TGF-β Signaling
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Arsenic trioxide (Sigma-Aldrich) was prepared in 1 N NaOH at 250 mM and then diluted in sterile water for a stock concentration of 1 μM. Cell culture medium, FGM-2 and DMEM, were purchased from Lonza (Allendale, NJ) and Gibco. Human recombinant TGF-β1 was purchased from R&D systems (Minneapolis, MN). Antibodies used were: alpha-SMA (Sigma-Aldrich, 1:10,000), type-1 collagen (abcam, 1:2000), PML (Santa Cruz, 1:500), PAI-1 (peprotech, 1:2000), fibronectin (BD science, 1:500). Antibodies for Smad2, p-Samd2, Smad3, p-Smad3, Akt, p-Akt, Erk, p-Erk, p38, p-p38 were purchased from Cell signaling and used at a concentration of 1:1000.
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