We used Proteo-JETTM Mammalian Cell Lysis Reagent (Thermo) to lyse cells. Crude lysates were centrifuged at 14,000g for 20 min at 4 °C. Lysate proteins were mixed with loading buffer and denatured by heating at 100 °C for 10 min. Proteins were resolved by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA), blocked with 5% nonfat milk, and then incubated with primary antibodies overnight at 4 °C on a shaking table. Primary antibodies targeting E-cadherin (1:1000), Vimentin (1:1000), AKT (1:1000), AKT-pS473 (1:1000), AKT-pT308 (1:1000), ERK (1:1000), p-ERK (1:1000) (Cell Signaling Technology, Danvers, MA, USA), α-SMA (1:1000), TBK1 (1:1000) (Abcam, Cambridge, MA, USA), and GAPDH (Beyotime, China) were used. Then, the blots were incubated with the secondary antibody, HRP-conjugated IgG (Beyotime, dilution 1:5000), for 1 h at room temperature. Specific bands for each protein were detected with an Image Quant LAS4000 imager (GE Healthcare Life Science, Pittsburgh, PA) using Amersham ECL (Millipore). Densitometric analysis of expression was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The expression of each molecule was normalized to GAPDH expression.
Imagequant las 4000 imager
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden, Spain, Canada
The ImageQuant LAS 4000 is a digital imaging system designed for analysis of protein and nucleic acid gels and blots. It captures high-quality images using a charge-coupled device (CCD) camera and provides quantitative data for a variety of applications, including Western blotting, EMSA, and Northern and Southern blotting.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (107 mentions)
Imagequant tl software, by GE Healthcare (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (5 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (5 mentions)
Imagequant tl, by GE Healthcare (5 mentions)
Hybond, by Cytiva (5 mentions)
Bca assay, by Thermo Fisher Scientific (5 mentions)
Clarity western ecl substrate, by Bio-Rad (5 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (4 mentions)
Amersham ecl western blotting, by Cytiva (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Amersham ecl prime, by Cytiva (4 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (4 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (4 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (4 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (3 mentions)
146 protocols using imagequant las 4000 imager
1
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
2
Western Blot Analysis of B. pertussis Virulence Factors
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B. pertussis Tohama I and its variants lacking expression of FHA and/or PT were cultured for 20 h at 37 °C in liquid SS medium, and cells from 1 mL of the bacterial culture were collected by centrifugation (10 min, 15,000× g). The pelleted cells were lyzed in 100 µL of TUS buffer (50 mM Tris-HCl (pH 8.0), 8 M urea, 2% (w/v) SDS), mixed with 50 µL of Laemmli buffer (50 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% glycerol (v/v), 0.1% (w/v) bromophenol blue, 1% (v/v) β-mercaptoethanol) and heated at 100 °C for 5 min. Whole bacterial cell lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Pall Corporation, Pensacola, FL). The membrane was blocked with 5% BSA in PBS for 2 h at RT and incubated with a mouse mAb specific to the S1 subunit of PT (diluted 1:500) or with mouse anti-FHA F1 mAb (diluted 1:100) for 1 h at RT. After washing with PBS, the membrane was incubated with HRP-conjugated anti-mouse IgG antibody (diluted 1:5000) for 1 h at RT and washed with PBS. Blots were developed using SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific, Rockford, IL, USA) and the chemiluminescent signal was recorded using an ImageQuant LAS 4000 Imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
3
Quantifying Barley Oxalate Oxidase Using Western Blotting
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Samples were loaded into precast 4–20%SDS-PAGE gels (BioRad) and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline, pH 7.4 containing 0.05% Tween 20 (PBST), at room temperature for 1 h, and were incubated with primary anti-barley oxalate oxidase 1 antibody (Origene, AP21356AF-N, rabbit polyclonal, 1:200 dilution) at 4°C overnight. Membranes were washed with PBST and a secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP; Cell Signaling Technology, #7074, 1:1000 dilution) was incubated at room temperature for 1 h, and then washed with PBST. HRP signal was activated using SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and Images were taken using an ImageQuant LAS 4000 imager (GE Healthcare). The antibodies were diluted to their appropriate ratio according to manual instructions. Experiments were performed in triplicate and a representative blot was shown. Original uncropped and unadjusted blot and gel images are shown in S1 File .
4
Comprehensive Protein Analysis of PRC2 Complex
5
Western Blot Analysis of Protein Expression
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Liver and small intestine tissues were homogenized in a lysis buffer containing a phenylmethylsulfonyl fluoride (Roche, Mannheim, Germany) and protease inhibitor cocktail (Sigma, Deisenhofen, Germany) to prepare protein lysates. Protein were separated in a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to nitrocellulose membranes (GE healthcare, Piscataway, NJ, USA). The membranes were blocked with 5% BSA and 0.1% Tween-20 in PBS solution for 1 h at room temperature with constant agitation. The membranes were then probed with the primary antibodies listed in Table S1 , followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA). The protein bands were visualized using enhanced chemiluminescence reagents on an ImageQuant LAS-4000 imager (General Electric, Pittsburgh, PA, USA) and quantified using the ImageJ software (NIH).
6
Detecting Overexpressed Transcription Factors and Secreted Cytokines in AF-MSCs
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To detect overexpressed transcription factors and secreted cytokines in AF-MSCs, 3 × 105 cells were plated on 100 mm plates and cultured in proliferation media for 48 h. In the case of tissue samples for specific hair regrowth markers, the obtained tissues were ground using a homogenizer. Total protein was extracted with RIPA buffer, and the mixture was centrifuged at 12,000 × g for 30 min at 4 °C. For normalization of relative expression, protein concentrations were measured using the Bradford Assay Kit (Bio-Rad). Extracted proteins were separated on precast 4–12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Invitrogen) by electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). For immunological reactions, membranes were incubated with the indicated primary antibodies at 4 °C overnight. Primary antibodies are listed in Supplemental Table 2 . Blots were incubated with secondary antibodies (horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG) for 1 h at room temperature and then imaged using an ECL Kit (Pierce, Rockford, IL, USA) on an ImageQuant LAS 4000 imager (GE Healthcare Life Sciences, Chicago, IL, USA).
7
Detecting Endothelial Markers in Kidney Tissue
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Protein was extracted from kidney samples on ice by homogenizing the tissue in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease inhibitors. Insoluble constituents were removed by centrifugation for 10 min at 4°C, at maximum speed. For Western blot analysis, 50 µg protein was subjected to SDS-PAGE on a precast gradient gel (Bio-Rad, Hercules, CA, USA) and transferred to a PVDF membrane (MilliporeSigma, Burlington, MA, USA) by wet blotting. After blocking for 1 h with 5% BSA in Tris-buffered saline with 0.05% Tween20 (TBS-T), membranes were incubated overnight at 4°C with rat anti–panendothelial cell antigen IgG2a antibody (MECA-32; Santa Cruz Biotechnology, Dallas, TX, USA), diluted 1:500 in 5% BSA in TBS-T. After washing in TBS-T, membranes were incubated for 1 h at room temperature with rabbit anti-rat horseradish peroxidase (Agilent Technologies, Santa Clara, CA, USA), diluted 1:10,000 in 0.5% BSA in TBS-T. Membranes were washed twice in TBS-T and once in Tris-buffered saline, incubated for 5 min with chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific) and visualized on an ImageQuant LAS 4000 Imager (GE Healthcare, Waukesha, WI, USA).
8
Recombinant SARS-CoV-2 Protein Expression
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293T cells were seeded in 10 cm dishes at a density of 3.5 million cells per dish. After overnight growth, cells were transfected using lipofectamine 3000 as described above. Plasmids pTwist-EF1alpha-nCoV-2019-S-2xStrep, pLVX-EF1alpha-nCoV-2019-E-IRES-Puro, pLVX-EF1alpha-nCoV-2019-M-IRES-Puro, or pLVX-EF1alpha-nCoV-2019-N-IRES-Puro were transfected using 90 μg of DNA per 10 cm dish. Cells were scraped 48 hours post-transfection, then lysed in RIPA buffer (EMD Millipore). Cell lysates were diluted with reducing Laemmli buffer, incubated for 10 minutes at 37°C, then ran on 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (BIO-RAD). Additionally, 1ug of purified recombinant S RBD-His6 was diluted in PBS and Laemmli buffer to a final volume of 20 μl and added to a 7.5% Mini-PROTEAN® TGX™ Precast Protein Gel (BIO-RAD). Resolved proteins were then transferred to a PVDF membrane, blocked in TBS with 2% BSA 0.1% Tween-20, then incubated with the following antibodies diluted to 1:500 in blocking buffer: mouse anti-SARS-CoV N monoclonal IgM 19c, mouse anti-SARS-CoV M monoclonal IgG1 283c, mouse anti-SARS-CoV E monoclonal IgM 472c, and mouse anti-2xStrep-tag antibody, and anti-His-HRP. Blots were stained with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) using an ImageQuant LAS 4000 imager (GE Life Sciences).
9
HIF-1α Expression in PC12 Cells
10
Protein Extraction and Analysis from Lung Tissue
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Lung tissues were homogenized in lysis buffer with protease inhibitor and phenylmethylsulfonyl fluoride to prepare protein lysates. Proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin solution for 1 h at room temperature. Then, the membranes were probed with primary antibodies overnight and incubated with peroxidase-conjugated secondary antibodies. The protein bands were visualized using enhanced chemiluminescence reagent on an ImageQuant LAS-4000 imager (General Electric, Pittsburgh, PA, USA) and quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).
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