Epididymal adipose tissue samples were homogenized with a polytron tissue homogenizer in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, and 1% Triton X-100) containing 5 mM iodoacetic acid and protease inhibitor (iNtRON Biotechnology, Inc., Seongnam, Korea), then solubilized at 4 °C for 40 min. Insoluble material was removed by centrifugation at 14,800× g for 10 min and the supernatant was collected and used for Western blot analyses. The protein contents of the lysates were measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Western blot analyses were conducted as described previously [37 (link)]. The antibodies against p-AMPKα (T172) (Cat No. 2535), AMPKα (Cat No. 2532), and β-actin (Cat No. 3700) were obtained from Cell Signal Technology (Beverly, MA, USA). Anti-rabbit IgG HRP-linked antibody (Cat No. 7074) and anti-mouse IgG HRP-linked antibody (7076) were obtained from Cell Signal Technology. Blots were detected with LuminataTM Forte Western HRP Substrate (Cat No. WBLUF0500, Millipore, Billerica, MA, USA). The relative protein abundances were determined using the ImageQuantTM LAS 500 imaging system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Protein expression levels were normalized to that of β-actin.
Imagequant las 500 imaging system
Manufactured by GE Healthcare
Sourced in Sweden, United States
The ImageQuant LAS 500 is a compact, high-performance imaging system designed for Western blot analysis and other gel-based quantification applications. The system uses a CCD camera and advanced software to capture and analyze images of chemiluminescent, fluorescent, and visible signals from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence method, by Bio-Rad (2 mentions)
Nuclear cytosol fractionation kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Anti rabbit igg hrp linked antibody cat no 7074, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti human igg, by Southern Biotech (1 mentions)
Nupage 4 12 gel, by Thermo Fisher Scientific (1 mentions)
West pico ecl reagent, by Thermo Fisher Scientific (1 mentions)
Instantblue, by Abcam (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
C myc, by GeneTex (1 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
Vascular endothelial growth factor (vegf), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Proteome profiler antibody array kit, by R&D Systems (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
16 protocols using imagequant las 500 imaging system
1
Epididymal Adipose Tissue Protein Analysis
2
Cytokine Profiling of LPS and HFPGE Exposure
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Cells were treated with LPS (0.1 μg/mL) or HFPGE (0, 5, 10 or 50 μg/mL), after which the 24-h conditioned media were collected as described above. The 24-h conditioned media were thereafter applied to a proteome profilerTM mouse cytokine array kit to investigate changes in the expressions of cytokines subsequent to LPS or HFPGE exposure, in accordance with the manufacturer’s instructions. The relative abundance of each protein spot was quantified using an ImageQuantTM LAS500 imaging system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The expression levels were normalized to the reference protein spots, after which the levels of TNF-α, IL-1β, IL-6, CXCL10, G-CSF, GM-CSF, and MCP-1 were measured using the relevant ELISA kits, following the manufacturer’s instructions.
3
Antibody Characterization by SDS-PAGE and Western Blot
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Two micrograms of purified antibodies were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS – PAGE) under non-reducing conditions with NuPage® 4–12% gel (ThermoFisher Scientific, MA, USA). For reducing conditions, the antibodies were reduced with a buffer containing 10 mM DTT. The protein bands were then visualized by InstantBlue™ (Abcam, Cambridge, UK) staining. For Western blot analysis, the proteins on SDS-PAGE gel were transferred into nitrocellulose membrane (Bio-Rad, Hercules, USA) and blocked with 5% BSA in PBS before probing with 1:5000 HRP-conjugated goat anti-human IgG (Southern Biotech, USA) or otherwise as stated in the figure. The membranes were then developed and visualized by chemiluminescence using West Pico ECL reagent (ThermoFisher Scientific, MA, USA) and visualized by the ImageQuantTM LAS500 imaging system (GE Health care Uppsala, Sweden).
4
Lung Protein Expression Analysis
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Lung homogenate proteins were isolated using a Nuclear/Cytosol fractionation kit (Thermo Fisher Scientific Inc, Bartlesville, OK, USA). Equal amounts of lysate protein were separated by 8–15% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% skim milk. They were then incubated with primary antibodies against Proliferating cell nuclear antigen (PCNA), Glucose transporter 1 (GLUT1), (Santa Cruz Biotechnology, Inc., TX, USA.), Carbonic anhydrase 9 (CA9) (GeneTex, Inc. CA, USA.), c-Myc, cyclin D1, cyclin-dependent kinase 4 (CDK4), survivin, p21, HIF-1α, VEGF, Bcl2, mammalian target of rapamycin (mTOR), ERK1/2, β-catenin, the nuclear factor erythroid 2-related factor 2 (Nrf2), or β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA) at a 1:1,000 dilution at 4 °C overnight. Target proteins were detected using the enhanced chemiluminescence method (Bio-Rad, Hercules, CA, USA). Signal detection was performed using ImageQuant LAS 500 Imaging System (GE Healthcare, Life Sciences, Korea). Collagenolytic activities in serum were measured by gelatin zymography. The gels were stained with Coomassie Brilliant Blue R-250 and then destained with methanol-acetic acid-water.
5
Androgen Receptor Immunoblotting in Mouse Testes
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Testes from WT, Inpp4b-/- and cryptorchid Rxfp2-/- mice were homogenized with a glass tissue grinder in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors [23 (link)]. The lysates were diluted tenfold and 20–30 μg of protein were resolved on SDS-PAGE and transferred to PVDF membrane. For immunoblotting, rabbit polyclonal primary antibody against AR (1:1000 dilution, catalog number 06–680, Millipore, Carlsbad, CA) and mouse monoclonal β-tubulin (1:5000, #05–661, Millipore) were used. Signal was visualized using ImageQuant LAS 500 imaging system (GE Healthcare Life Sciences, Marlborough, MA) and quantitative analysis was performed with ImageQuant TL software.
6
Proteomic Analysis of Angiogenesis Factors
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Conditioned media and cell lysates were analyzed by a Proteome Profiler™ Antibody Array kit (R&D Systems, Minneapolis, MN, USA) to estimate the expression levels of various angiogenesis (metastasis)-related proteins, according to the manufacturer's instructions. The relative abundance of each protein spot was quantified using an Image Quant™ LAS 500 imaging system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The expression levels were normalized to the control protein.
7
Fisetin Mitigates PM2.5-Induced Stress
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HaCaT keratinocytes were treated with fisetin (0–10 μM) for 2 h prior to exposure to 100 μg/mL PM2.5 for 24 h. The cells were subsequently lysed with Radioimmunoprecipitation Assay Buffer (RIPA) (iNtRON Biotechnology, Seongnam, Gyeonggi-do, Korea) with protease inhibitors (Sigma-Aldrich) and the proteins were quantified using Bio-Rad Protein Assay Reagents (Bio-Rad, Hercules, CA, USA). Western blotting was performed, and the protein expression was quantified using an ImageQuant LAS 500 Imaging System (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). β-Actin was used as the loading control.
8
Immunoblotting and Quantification of Cell Signaling Proteins
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Cell extracts were prepared in Laemmli sample buffer. Protein concentrations were determined using Bradford assays. Proteins were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Blots were blocked with 5% dry milk in 0.1% Tween-20 in PBS, incubated with primary antibodies, followed by HRP-conjugated secondary antibodies. The following primary antibodies and dilutions were used: eIF3e (1:1000, C-20, described previously, [52 (link)]), β-actin (1:4000, A5441, Sigma-Aldrich), PARP1 [1:1000, 9532, Cell Signaling Technology (CST)], PARP2 (1:1000, NBP2-47337, Novus Biologicals), PAR (1:1000, ALX-804-220-R100, Enzo Life Sciences), Lys48-linked ubiquitin conjugates (1:1000, 05-1307, Millipore), S6 (1:1000, 2217, CST), pS6 (1:2000, 4858, CST), S6K1 (1:1000, 9202, CST), pS6K1 (1:1000, 9234, CST), TSC2 (1:1000, 4308, CST), 4EBP1 (1:1000, 9452, CST), p4EBP1 (1:1000, 9451, CST). Membranes were developed using ECL Prime reagent and scanned with an ImageQuant LAS500 imaging system (GE Healthcare). Bands of interest were quantified with the ImageQuant TL software (GE Healthcare).
9
Protein Expression and EMSA Analysis
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The expression and purification of proteins, including DNA-PKcs-B WT and DNA-PKcs-B K525R/N, Ku70, and Ku80, were accomplished in BL21 E. coli. Subsequently, EMSA assays were conducted using the EMSA Kit (Thermo Fisher #20148) following the manufacturer’s instructions. Briefly, proteins at specific concentrations were blended with biotin-labeled DNA (2 fM) substrate in a 20 µL reaction mixture (5 mM MgCl2, 2.5% glycerol, 50 ng/µL poly (dI.dC), 0.05% NP-40). The samples were subsequently subjected to a 20-min incubation at room temperature. Finally, the samples were separated on 6% native polyacrylamide gels in 0.5× TBE buffer (2 mM EDTA, 90 mM boric acid, 90 mM Tris-HCl, pH 8.3) and detected with an HRP-conjugated biotin antibody using a GE ImageQuant™ LAS 500 Imaging System.
10
TAS2R50 Protein Expression in NCI-H716 Cells
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NCI-H716 cells (1 × 106 cells/well) were seeded onto pre-coated 6-well plates and incubated at 37 °C under 5% CO2 for 48 h. After starvation, the cells were treated with ISS for 1 h. The NCI-H716 cells were lysed with cell lysis buffer (Cell Signaling, Danvers, MA, USA). Proteins were separated by SDS-PAGE (10%) and transferred to a PVDF membrane. The membrane was blocked with 3% bovine serum albumin (BSA) and incubated with primary antibodies against TAS2R50. The TAS2R50 polyclonal antibody and β-actin were purchased from Bioss Antibodies (Woburn, MA, USA) and Santa Cruz (Dallas, TX, USA), respectively. The protein expression levels were developed using an enhanced chemiluminescence (ECL) detection system and detected using an ImageQuant LAS 500 imaging system (GE Healthcare Life Sciences, New Zealand, NSW, Australia). The intensities of the individual bands were calculated using ImageJ software (NIH, New York, NY, USA).
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