MNT-1 cells were cultured in 6-well plates for 24 h then treated with L-anserine, L-carnosine (1 and 5 μg/mL), and ACCE (1.0 × 104 and 2.0 × 104 μg/mL) for 24 or 72 h.; cells treated with PBS were used as a control. The cells were washed with PBS and lysed in cell lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.4) containing protease inhibitor and phosphatase inhibitor (Roche, Mannheim, Germany). The cell lysates were run on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore; Darmstadt, Germany). Non-specific reactivity was blocked with 5% skim milk in PBS for 1 h. The membranes were then probed with the given concentration of specific primary antibodies; 1:1000 anti-Akt, 1:1000 anti-pAkt (S473), 1:1000 anti-ERK, 1:1000 anti-pERK (T202/Y204) (Cell Signaling Technology; Danvers, MA), 1:2000 anti-tyrosinase (Invitrogen, Carlsbad, CA), and 1:10,000 anti-β-actin (Sigma Aldrich, St. Louis, MO, USA). The signals were visualized using the ECLTM Prime Western Blotting Detection System (AmershamTM, Buckinghamshire, UK) and detected using the ImageQuant LAS 4000 mini-image analyzer and ImageQuantTM TL analysis software (GE Healthcare, Buckinghamshire, UK).
Imagequant tl analysis software
Manufactured by GE Healthcare
Sourced in United Kingdom
ImageQuant TL analysis software is a digital imaging and analysis tool designed for use with GE Healthcare's life sciences products. It provides quantitative analysis of images obtained from various imaging techniques, such as gel electrophoresis, Western blotting, and protein gels.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las4000 mini image analyzer, by GE Healthcare (4 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Coomassie brilliant blue g 250, by Bio-Rad (2 mentions)
Lowry dc protein assay, by Bio-Rad (2 mentions)
Typhoon imager, by GE Healthcare (2 mentions)
Bodipy iam, by Thermo Fisher Scientific (2 mentions)
Bodipy iam, by GE Healthcare (2 mentions)
Protease and phosphotase inhibitors cocktail, by Roche (2 mentions)
Imagequant las 4000 mini biomolecular imager, by GE Healthcare (2 mentions)
Ecl select western blotting detection kit, by GE Healthcare (2 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti pakt s473, by Cell Signaling Technology (1 mentions)
Anti p erk t202 y204, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Luminata crescendo, by Merck Group (1 mentions)
22 protocols using imagequant tl analysis software
1
Investigating Anserine and Carnosine Effects
2
Cell Extract Immunoblotting Protocol
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Total cell extracts were prepared as previously described [64 (link)]. Primary and secondary antibodies used for immunoblotting are reported in Table S4 . Band intensities were measured by means of ImageQuant™ LAS 4000 Chemiluminescence Camera System and ImageQuantTM TL analysis software (GE Healthcare Life Science, Chicago, IL, USA).
3
Analyzing GLI1 and GLI2 expression
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Cells at 70-75% confluence were treated with SMO antagonist compounds and cyclopamine at different concentrations (1 nM, 10 nM, 100 nM and 10 µM) for 24 hours. After treatment, cells were lysed with RIPA buffer and then disrupted by sonication for 5 minutes in an ice bath. Protein concentration was assessed according to Bradford. Forty micrograms of cells protein lysate were resolved by 8% SDS-PAGE and then proteins were electrotransferred onto nitrocellulose membrane (0.45 mm pore size; Whatman). After blocking for 45 minutes with 5% not-fat dry milk in 0.05% Tween-20 Tris buffer saline (TBS-T) at room temperature, membranes were incubated with anti-GLI1 (1:1000), anti-GLI2 (1:1000) and anti-GAPDH (1:50.000) primary antibodies at 4°C overnight. The membranes were then washed three times with TBS-T and then incubated with 1:80.000 dilution of anti-rabbit HRP-conjugated secondary antibodies. Immunoreactive bands were detected using Luminata Crescendo (Millipore) and images were acquired using ImageQuant LAS4000 (GE Healthcare). The optical densities of the bands were analysed by ImageQuant TM TL analysis software (GE Healthcare, RRID: SCR_014246) using GAPDH as a loading normalizing factor. The experiment was performed in triplicate.
4
Northern Blot Analysis of Transcripts
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Cell fractionation was performed as described previously (20 (link)). Total RNA was extracted (Nucleospin RNAII; Takara Bio) and poly(A)+ RNA was purified (NucleoTrap mRNA Mini kit; Takara Bio) according to the manufacturer's instructions. The RNA samples were loaded on 1.5% gels (NorthernMax-Gly Kit; Life Technologies), transferred to Hybond N+ nylon membranes (GE Healthcare) and probed with internally DIG-labeled sequences following pre-hybridization in ULTRAhyb hybridization buffer (Ambion). DIG-labeled RNA probes were prepared from template DNA amplified by specific primers (Supplementary Table S2) using a DIG Northern Starter Kit (Roche). Visualization of transcripts was performed with a CDP-Star reagent (Roche). Signals were detected by an LAS4000 mini biomolecular imager (GE Healthcare). The densitometric analysis of each band was performed by ImageQuant TL analysis software (GE Healthcare).
5
Protein Expression Analysis in CCA Cells
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Total protein was extracted from CCA cell lines using 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH7.4 containing phosphatase inhibitor, protease inhibitor, and 5 μM PUGNAc. Protein (30 μg) was solubilized in the sample loading medium (SM; 62.5 mM Tris-HCl pH 6.8, 2% SDS, 5% β-ME, 10% Glycerol, and 0.01% Bromphenol blue), separated by SDS-PAGE according to Laemmli38 (link), and electro-transferred onto a PVDF membrane using Bolt and Marhoney transferring buffer (40 mM Tris base, 20 mM sodium acetate, 2 mM EDTA, pH 7.4, 20% methanol, and 0.05% SDS)39 (link). Immunodetection of particular proteins was performed using specific monoclonal antibodies as follow; 1:200 of anti-OGT and anti-MMP7 (Santa Cruz Biotechnology, Santa Cruz, CA); 1:400 of anti-β-catenin (BD Transduction Laboratories, New Jersey); 1:1000 of anti-NF-κB (p65) (Santa Cruz), anti-β-tubulin (Santa Cruz, CA), anti-O-GlcNAc (RL2, Pierce Biotechnology, IL), anti-Akt and anti-pAkt (Cell Signaling Technology, Danvers, MA). The immunoreactivity was detected with the ECLTM Prime Western Blotting Detection System (AmershamTM, Buckinghamshire, UK) and analyzed using an ImageQuant LAS 4000 mini image analyzer and ImageQuant™ TL analysis software (GE healthcare, Buckinghamshire, UK).
6
Fluorescent detection of protein thiols
7
Quantifying Muscle Regulatory Proteins
8
Western Blot Quantification and Analysis
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CCA cells were lysed in NP-40 lysis buffer18 (link). Protein concentration was determined using the Bradford reagent (Bio-rad laboratories, Hercules, CA). Protein lysate was separated on SDS-PAGE and transferred onto a PVDF membrane. The immunoreactivity was detected using the ECLTM Prime Western Blotting Detection System (GE Healthcare). The signals were captured by the ImageQuant™ LAS 4000 mini-image analyzer and the signal intensities were analyzed by ImageQuant™ TL analysis software (GE Healthcare).
9
EGF-Induced Signaling Pathway Analysis
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After different drug treatment, the cells were stimulated with 10 ng/ml EGF for 15 min before harvesting, washed twice with cold PBS and lysed with RIPA buffer that containing protease and phosphotase inhibitors cocktail (Roche, UK). The supernatants were collected after centrifugation at 12000 rpm for 20 min. The protein was applied to polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and then detected by the proper primary and secondary antibodies before visualization with a chemiluminescence kit. The intensity of blot signals was quantitated using ImageQuant TL analysis software (General Electric, UK).
10
Fluorescent detection of protein thiols
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To detect protein thiols, Bodipy-IAM (Bodipy-iodoacetamide; Invitrogen, Eugene, OR), a fluorescently labeled alkylating agent capable of forming covalent adducts with cysteine thiol groups that are not involved in disulfide linkages was utilized. In brief, PCF samples from individual patients (0 and 4 h post-surgery) were diluted 1:10 with de-ionized water prior to determining protein concentration using the Lowry DC protein assay (Bio-Rad). Subsequently, 5 μg of PCF protein was treated with 500 μM Bodipy-IAM for 30 min to assess protein thiol modifications. The reaction was stopped with 5X SDS-PAGE sample buffer (1M Tris-HCl, pH 6.8, 10% SDS, 30% glycerol, 0.05% bromophenol blue) containing 5% β-mercaptoethanol.
Samples were resolved using 12.5% SDS-PAGE gels and imaged in-gel using a Typhoon imager (GE Healthcare Biosciences, Pittsburgh, PA). Following imaging, gels were immediately stained with Coomassie Brilliant Blue G-250 (coomassie blue; Bio-Rad Laboratories, Hercules, CA) for 1 hour, destained overnight, and imaged using the AlphaView SA imager (Protein Simple, Santa Clara, CA) to evaluate protein loading. The Bodipy-IAM fluorescent signal intensity for albumin was quantified using ImageQuantTL analysis software (GE Healthcare Biosciences, Pittsburgh, PA) and the coomassie blue protein stain for albumin was quantified using the AlphaView SA software.
Samples were resolved using 12.5% SDS-PAGE gels and imaged in-gel using a Typhoon imager (GE Healthcare Biosciences, Pittsburgh, PA). Following imaging, gels were immediately stained with Coomassie Brilliant Blue G-250 (coomassie blue; Bio-Rad Laboratories, Hercules, CA) for 1 hour, destained overnight, and imaged using the AlphaView SA imager (Protein Simple, Santa Clara, CA) to evaluate protein loading. The Bodipy-IAM fluorescent signal intensity for albumin was quantified using ImageQuantTL analysis software (GE Healthcare Biosciences, Pittsburgh, PA) and the coomassie blue protein stain for albumin was quantified using the AlphaView SA software.
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