Growth-arrested cells were incubated with different treatments for the indicated time intervals. The cells were either untreated or treated with quercetin or inhibitors of TNF-α antagonist, c-Src (PP1), ERK1/2 (U0126), c-Fos (TSIIA), or NF-κB (BAY). After treatment, the cells were washed with PBS, scraped, lysed in lysis buffer, and collected as a whole-cell extract at 4 °C using a previously described standard protocol[42 (link)]. Samples were loaded onto a 10% SDS-PAGE gel, electrophoresis was performed, and the bands were transferred to a PVDF membrane. The PVDF membrane was incubated overnight with specific primary antibodies, including anti-phospho-c-Src, anti-phospho-ERK1/2, anti-phospho-c-Fos, anti-phospho-NF-κB, or anti-GAPDH. The PVDF membranes were washed and then incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (dilution, 1:2000) for 1 h. The relative expression levels of c-Src, ERK1/2, c-Fos, and NF-κB were normalized to that of GAPDH. Finally, the immunoreactive bands were incubated in enhanced chemiluminescence reagent, and images were recorded on a UVP BioSpectrum 500 Imaging System (UVP, Inc.; Upland, CA, United States). Densitometry was quantified using UN-SCAN-IT gel software (Silk Scientific, Inc.; Orem, UT, United States).
Un scan it gel software
Manufactured by Silk Scientific
Sourced in United States
UN-SCAN-IT gel software is a data analysis tool designed to quantify and analyze gel images, such as those from electrophoresis experiments. The software provides functionality to measure band size, intensity, and other parameters from uploaded gel images.
Automatically generated - may contain errors
Lab products found in correlation
Acetic acid, by Merck Group (4 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Amersham hybond p transfer membranes, by GE Healthcare (2 mentions)
Ecl plus western blot detection system reagents, by GE Healthcare (2 mentions)
Hybond, by Cytiva (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Chemiluminescence detection system, by Thermo Fisher Scientific (2 mentions)
Anti rabbit or anti mouse igg conjugated to horse radish peroxidase, by Cell Signaling Technology (2 mentions)
Cyclophilin b, by Abcam (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Autoradiography film, by Kodak (2 mentions)
Cdp star, by Merck Group (2 mentions)
Uvp biospectrum 500 imaging system, by Analytik Jena (1 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Total oxphos cocktail, by Abcam (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
33 protocols using un scan it gel software
1
Western Blot Analysis of Signaling Pathways
2
Western Blot Analysis of Cellular Signaling
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Proteins were loaded into 10% polyacrylamide gels. Protein transferred to nitrocellulose membranes, which were immunoblotted with antibodies against AMPKα, p-AMPKα, ACC, p-ACC, NF-κB p65, p-NF-κB p65, ac-NF-κB p65, and SIRT1. Protein bands were visualized using the Supersignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA). Signals from bands were quantified using a UN-SCAN-IT gel™ software (Silk Scientific, Orem, UT, USA).
3
Western Blot Analysis of Liver Samples
4
Western Blot Quantification Protocol
5
Differential Transcription Factors in ApoE-Mediated Alzheimer's
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NRA cells were treated with a combination of A or scrambled control and recombinant ApoE isoforms. Nuclear material was isolated using a kit purchased from Panomics Inc. (Santa Clara, CA, USA). Protein concentration of each sample extract was then determined by BCA protein assay. The Protein/DNA Combo array was purchased from Panomics Inc. (Santa Clara, CA, USA). Hybridization, probe binding and detection were performed according to manufacturer's instructions. Blots were analyzed using UN-SCAN-IT gel software (Silk Scientific, Inc, Orem, UT, USA). Three steps were used to identify the TFs of interest. The initial analysis identified the TFs that showed at least a 2-fold change in intensity between ApoE2 + A and ApoE3 + A treatments. The second processing step identified TFs that also did not change between the ApoE2 + A scrambled and ApoE3 + A scrambled treatments. The last screening step identified those TFs that also changed between the A relative to scrambled treatments. This yielded 7 TFs that were upregulated and 36 TFs that were downregulated with ApoE2 + A treatment as compared to ApoE3 + A treatment (Table 2). This list was narrowed even further by listing only those TFs that had a reported link to either AD and/or inflammation (Table 3).
6
Quantitative Analysis of Calcium Channels
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Fresh full-thickness cortical brain tissue was dissected from saline-treated UC and BD-treated UC cortical areas and sonicated in homogenization buffer: 12.5% 0.5 M Tris-HCl, 10% glycerol, 2% SDS and protease inhibitor cocktail (Thermo Scientific). Homogenates were centrifuged at 14 000g for 15 min at 4 °C and supernatant collected. Samples of protein (15–20 μg) were separated by 4–15% Tris-HCl running gel and transferred to Amersham Hybond-P transfer membranes (GE Healthcare). Membranes were blocked with 5% nonfat milk in TBST buffer for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against N-type calcium channel (Sy Sy Germany; 1:1000) and P/Q-type calcium channel (Sy Sy Germany; 1:1000). After several washes, the membranes were incubated with secondary antibodies at dilutions of 1: 20 000 for 1.5 h at room temperature. After further extensive washing, the immunoreactive bands were detected with ECL plus western blot detection system reagents (GE healthcare). Quantification of optical density (OD) of western blots was performed using Un-Scan-It gel software (v6.1, Silk Scientific). Relative expression of specific protein was normalized and calculated as the OD of specific protein/OD of β-actin.
7
Cardiac Tissue Western Blotting Protocol
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For Western blotting of cardiac tissue from the 1 week and 3 week groups, frozen tissue was crushed and lysis buffer added before tissue was homogenized; a protein assay established the protein concentration of each lysate. The same concentration of protein from each sample was loaded onto 12.5% SDS‐PAGE gels and was separated by electrophoresis.29 Primary antibodies for PDK 1 and 2 were purchased from New England Biolabs and Abgent, respectively; an antibody for PDK4 was kindly donated by Prof. Mary Sugden (Queen Mary's, University of London, UK). A primary antibody for LDH was purchased from Abcam (ab52488). Even protein loading and transfer were confirmed by Ponceau staining (0.1% w/v in 5% v/v acetic acid, Sigma‐Aldrich), and internal standards were used to ensure homogeneity between samples and gels. Bands were quantified using UN‐SCAN‐IT gel software (Silk Scientific, USA) and all samples were run in duplicate on separate gels to confirm results.
8
Myosin Expression Quantification in Drosophila
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Relative levels of myosin expression were determined by measuring myosin-to-actin ratios in upper thorax homogenates subjected to one-dimensional SDS polyacrylamide gel electrophoresis (O'Donnell et al., 1989 (link)). Six dissected upper thoraces from 2-day-old adult female flies for each line were homogenized in 360 μl of SDS gel 1× Laemmli Sample Buffer (Bio-Rad, Hercules, CA) containing 5% 2-mercaptoethanol. Samples were boiled for 5 min prior to loading at 2-5 μl on a 15-well 10% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad). Gels stained with Coomassie Brilliant Blue R-250 were digitally scanned using an Epson Expression 636 flatbed scanner. Myosin heavy chain and actin levels were determined by UN-SCAN-IT gel software (Silk Scientific) for three separate lanes per sample. The median myosin-to-actin ratio in mutant thoraces was compared with the median ratio found in thoraces from aged-matched flies expressing the PwMhc2 control transgene in the corresponding genetic background.
9
Western Blot Analysis of Cell Signaling Proteins
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Whole cells were lysed in a lysis buffer [20 mM Tris-HCl (pH 8.0), 1% nonyl phenoxypolyethoxylethanol-40, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 50 mM NaF, and 1 mM Na3VO4] supplemented with a protease inhibitors cocktail (Roche Applied Science, Indianapolis, IN, USA). After incubation on ice, lysed tissues were centrifuged, and equal amounts of protein were dissolved in 4× sodium dodecyl sulfate (SDS) sample buffer. Samples were separated in 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and blocked with 5% nonfat dried milk. The membrane was incubated with the primary antibody (1:1,000 dilution) at 4°C overnight. Antibodies against p53, p21, Cdk4, Cyclin D1, Cdk2, Cyclin E1, Bax, Bcl-2, Caspase-3, and β-actin were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The membrane was washed with Tris-buffered saline with Tween 20, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc.). Proteins were detected using the chemiluminescence (Western ECL substrate, BioRad). Membrane images were recorded using a ChemiDoc XRS system (BioRad). Protein bands were quantitated by densitometry using the UN-SCAN-IT gel software (Silk Scientific, Orem, UT, USA).
10
Western Blotting Analysis of Cell Lines
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For western blotting analysis, cells (T98 G or H460 cell lines)) or tissues (H460 cells) were harvested and then washed with ice-cold phosphate-buffered saline (PBS) twice before the addition of RIPA lysis buffer containing protease and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Protein concentration was quantified using the bicinchoninic acid protein assay. Equal amounts of protein (20 μg per lane) were loaded into each well and separated by 7.5 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer onto polyvinylidene difluoride membranes (Bio-Rad, Richmond, CA, USA). Membranes were blocked using 5% nonfat dry milk or 2% bovine serum albumin (in PBS with 0.05% tween-20. The blots were then incubated with primary antibodies (1:200–1:1000) overnight at 4 °C. Secondary antibody anti-rabbit or anti-mouse IgG conjugated to horse radish peroxidase (1:5000; Cell Signaling Technology) was incubated for 2 h at room temperature. Immunoblots were developed using the chemiluminescence detection system (Thermo Scientific, Waltham, MA, USA). Quantification of immunoblotting signal of respective proteins was performed using the UN-SCAN-IT Gel software (Silk Scientific, Inc., Orem, UT, USA).
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