Cell lysates were prepared by suspending 5 × 106 Jurkat T cells in 300 μl of lysis buffer as described elsewhere [49 ]. Equivalent amounts of protein lysate (20 μg) were electrophoresed on a 4-12% NuPAGE gradient gel (Invitrogen/Novex, Carlsbad, CA, USA) and then electrotransferred to an Immobilon-P membrane. Protein detection was performed using an ECL western blot kit according to the manufacturer's instructions. Densitometry was performed using ImageQuant TL software (Amersham, Arlington Heights, IL, USA). The arbitrary densitometric units for each protein of interest were normalized using those for GAPDH or β-actin.
Imagequant tl software
Manufactured by Cytiva
Sourced in United States, Germany
ImageQuant TL software is a computer-based image analysis tool designed for quantitative analysis of images from gel electrophoresis, Western blots, and other gel-based applications. The software provides tools for image capture, band/spot detection, and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Amersham typhoon, by Cytiva (7 mentions)
Ecl plu, by Cytiva (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Amersham typhoon 5 scanner, by Cytiva (3 mentions)
Typhoon 9400 variable mode imager, by Cytiva (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Las 4000, by Cytiva (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Ab27649, by Abcam (2 mentions)
A5060, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Amersham typhoon scanner, by Cytiva (2 mentions)
Myecl imager, by Thermo Fisher Scientific (1 mentions)
Myimageanalysis software, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti actin c4, by MP Biomedicals (1 mentions)
Ecl western blot kit, by Cytiva (1 mentions)
Human cytokine antibody array, by RayBiotech (1 mentions)
55 protocols using imagequant tl software
1
Western Blot Protein Analysis
2
Vascular Signaling Pathway Analysis
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Aortic inflammation and vascular insulin and VEGF signaling were analyzed by performing Western blots to examine IκBα degradation, a marker of NF-κBα activation, and insulin or VEGF-induced Akt phosphorylation (17 (link)–20 (link), 31 (link)). In addition, IκBα degradation was examined to test for the activation of the NF-κBα pathway by FFA in HUVECs. As described before (17 (link)–20 (link), 30 (link), 31 (link)), tissue and cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Bio-Rad, Hercules, CA), and probed with antibodies against IκBα and actin or phospho-Akt (Ser473) and Akt. Antibodies against IκBα, phospho-Akt (Ser473), and Akt (1:1,000) were obtained from Cell Signaling Technology (Danvers, MA), and the actin (1:2,000) antibody was purchased from Sigma-Aldrich. The blots were developed with ECL plus (Amersham Biosciences, Piscataway, NJ; or Pierce, Thermo Fisher, Waltham, MA), and band intensities (Typhoon 9400 variable mode imager Amersham Biosciences; or myECL Imager, Thermo Fisher) were quantified using Image Quant TL software (Amersham Biosciences, Piscataway, NJ; or myImageAnalysis Software, Thermo Fisher).
3
PTEN Overexpression and Phospho-Akt Analysis
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Whole cell protein extracts from U2OS cells overexpressing ectopic PTEN and mutations were prepared by cell lysis in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% IGEPAL CO-630 (Nonidet P-40), 2 mM Na3VO4, 100 mM NaF, 1mM PMSF, 1 μg/ml of aprotinine, 20 mM Na4P2O7), followed by centrifugation at 15200 g for 10 min and collection of the supernatant. Yeast cell extracts were obtained by standard procedures. Proteins (50–100 μg in the case of mammalian cells; 20–50 μg in the case of yeast) were resolved in 10% SDS-PAGE under reducing conditions and transferred to PVDF membranes. Immunoblot was performed using anti-phospho-Ser473-Akt + anti-phospho-Thr308-Akt and anti-Akt antibodies (Cell Signaling Technologies), anti-PTEN 425A mAb (Andrés-Pons et al., 2005) or rabbit polyclonal anti-PTEN antibodies (Upstate), anti-GAPDH (Santa Cruz Technology) or anti-actin C4 (MP Biomedicals, France) antibodies, followed by horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse (Calbiochem) antibodies. For determination of phospho-Akt content, bands were quantified using ImageQuantTL software (Amersham Biosciences).
4
Western Blot Protein Detection
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Following cell lysate preparation, an equivalent amount of protein lysate (20–25 μg) was electrophoresed on a 4–12% NuPAGE gradient or 7% NuPAGE gels with MOPS buffer or Tris-Acetate Buffer and then electrotransferred to a nylon membrane as described elsewhere [8 (link)]. Protein detection was performed using an ECL western blot kit (Amersham, Arlington Heights, IL, USA). Densitometry was performed using ImageQuant TL software (Amersham) as previously described [34 (link)]. The arbitrary densitometric units for each protein of interest were normalized to the densitometric units for GAPDH.
5
Quantifying Antiviral Activity of CPT
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The assay is performed as reported elsewhere29 with some modifications. Cell-free virus particles were incubated with CPT at different concentrations at 37 °C in 5% CO2 for 3 h. The virus-drug mixture was then diluted 20-fold with 2% FBS DMEM to effective concentrations of CPT, after which the inoculum was added to MCF-7 cells seeded in 96-well plates (104 cells per well; final MOI 0.1). The cells were incubated at 37 °C in 5% CO2 for 3 days. After 3 days, the plates were scanned and analyzed for viral reporter fluorescence using the Typhoon 9410 variable mode imager (Amersham Biosciences; Baie d’Urfe, QC, Canada) to evaluate MV-EGFP infections. Fluorescence intensity was quantified using Image Quant TL software (Amersham Biosciences).
6
Human Cytokine Antibody Array Analysis
7
Colony Formation Assay for B78-H1 Cells
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B78-H1 cells have been treated with porphyrin/light and seeded in 60 mm Petri dish, at a density of 5 × 103 cells. After one week of growth, the colonies were fixed and stained with 2.5% methylene blue in 50% ethanol for 10 min. The images were obtained by Gel DOC 2000 Bio-Rad (Milan, Italy). The stained colonies (>50 cells) have been counted by an Image Scanner equipped with Image Quant TL software (Amersham Biosciences). The experiment was performed in duplicate.
8
Quantifying K+ Channel Subunits in Mice
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Corpus cavernosum and aorta from KCa2.3T/T (−Dox) mice, KCa2.3T/T (+Dox) mice and WT mice were snap frozen and kept at −80 °C in different quantities for KCa evaluation. Protein was extracted, quantified and mixed in sample buffer before samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The following protein amounts were used for detection: aorta 12 μg for KCa2.3; corpus cavernosum 12 μg for KCa2.3 (sc-28621), 10 μg for KCa3.1 or 8 μg for KCa1.1α (-alpha-subunit) (Alomone 1184–1200) or KCa1.1β1 (-beta-1-subunit) (ab3587); cerebellum and liver 8 μg for KCa1.1β1. Samples were incubated with the indicated antibodies, all raised in rabbits: KCa2.3 (1:200), KCa1.1α (1:400), KCa1.1β1 (1:500); and housekeeping proteins: pan-actin (1:1000) and beta-tubulin (1:200). Membranes were then incubated with a secondary anti-rabbit IgG (1:4000) and processed with an ECL-Plus kit (General Electric “GE” Health care). Bands were visualized with a luminescence camera (Image Quant LAS 4000 mini from GE) and intensity was quantified by Image Quant TL software (Amersham Biosciences).
9
Protein Expression Analysis in Tissues
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Protein was extracted by homogenizing ~100 mg of pulverized pituitary, arcuate nucleus of the hypothalamus and liver tissues in ice-cold lysis buffer using a polytron homogenizer. Homogenates were then centrifuged and the supernatants mixed with 2 × standard SDS sample loading buffer and then boiled at 95°C for 5 minutes. Approximately, 50 μg of protein extracts were separated in SDS-PAGE gels and transferred to nitrocellulose membranes for immunoblotting with primary antibodies for leptin, OB-Rb, GH releasing hormone (GHRH), GH inhibiting hormone (GHIH, somatostatin), GH receptor (GHR), IGF1, IGF-binding protein 3 (IGFBP3), IGF1 receptor(IGF1R), 11 β hydroxysteroid dehydrogenase (HSD)1, 11-β HSD2, Hexose-6-phosphate dehydrogenase (H6PD) and β-actin. The membranes were incubated with appropriate secondary antibodies and then visualized using enhanced chemiluminescence (Amersham Biosciences) and exposure to an X-ray film. The target protein band density was analyzed by ImageQuant TL software (Amersham Biosciences) and normalized according to the density of β-actin as previously described [30 (link)].
10
Liver Protein Expression Analysis
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The liver was lysed with 20 mM Tris buffer (pH 7.4) containing 2 mM EGTA, 137 mM NaCl, 1% NP40, 10% glycerol, and 12 mM α-glycerol phosphate and protease inhibitors. Lysates containing equal amounts of protein (30–50 μg) were used for immunoblotting with specific antibodies against protein kinase B (PKB/Akt), glycogen synthase (GSK)-3β, AMP kinase (AMPK), cAMP responding element binding protein (CREB), phosphoenolpyruvate carboxykinase (PEPCK), glucose transporter-2 (GLUT2), β-actin, phosphorylated forms of PKBSer473, GSK-3β, CREB, and AMPK (Cell Signaling, Danvers, MA), as previously described [16 (link)]. The intensity of protein expression was measured using Imagequant TL software (Amersham Biosciences, Piscataway, NJ).
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