De novo mitochondrial protein synthesis was assessed on exponentially growing cybrids in 12-well plates. Cells were washed twice in methionine-free DMEM (Sigma, St. Louis, MO, USA) and pulsed with 300 μCi/mL [35S]-methionine (PerkinElmer, Waltham, MA, USA) at 37 °C for 1 h in 300 μL of methionine-free DMEM supplemented with 10% dialysed FBS, emetine (100 µg/mL) and cycloheximide (100 µg/mL). After the radioactive pulse, cells were washed twice with phosphate-buffered saline (PBS) and dissolved in 1× Laemmli buffer. Aliquots (50 μg) of total cell protein were fractionated by 15% SDS-PAGE and radioactive signals were detected using the Typhoon FLA 9500 PhosphorImager and ImageQuant TL 8.1 software (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Post-detection protein loading was determined using Coomassie blue staining.
Imagequant tl 8
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden
The ImageQuant TL 8.1 software is a versatile tool for the analysis of gel-based and blot-based images. It provides a user-friendly interface for quantifying bands, spots, and other features within digital images, enabling researchers to obtain accurate and reproducible measurements. The software supports a wide range of input image formats and offers various analysis tools, such as lane detection, band quantification, and molecular weight calculation.
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Lab products found in correlation
Las 4000, by Cytiva (8 mentions)
Amersham typhoon, by Cytiva (8 mentions)
Imagequant las 4000, by GE Healthcare (6 mentions)
Amersham typhoon biomolecular imager, by GE Healthcare (5 mentions)
Amersham imager, by Cytiva (5 mentions)
Typhoon fla 7000 system, by GE Healthcare (3 mentions)
Typhoon fla 9500, by GE Healthcare (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Prism 8, by GraphPad (2 mentions)
Cay10006421, by R&D Systems (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Storage phosphor screen, by GE Healthcare (2 mentions)
Typhoon imaging system, by GE Healthcare (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Hybond, by Cytiva (2 mentions)
Typhoon 9400 scanner, by Cytiva (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Amersham imager 600rgb, by GE Healthcare (2 mentions)
Easytag, by PerkinElmer (2 mentions)
78 protocols using imagequant tl 8
1
Measuring Mitochondrial Protein Synthesis
2
Quantifying Telomeric Signals in Mouse Genomic DNA
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Mouse genomic DNA was analyzed as previously described [94 (link)]. The single-stranded telomere signals in the native gel were quantified with ImageQuant TL 8.1 (GE Healthcare Life Sciences, Marlborough, MA) software and the ratio of short versus bulk telomeric signals (regions denoted by boxes in Fig 1B ) are reported relative to cells expressing FokIDA-TRF1 (set at 100).
3
Western Blot Analysis of Stat3 Phosphorylation
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Western blotting was performed according to standard procedures. Proteins were extracted by using a double-strength SDS sample buffer, heated for 5 min at 90 °C, and stored at −80 °C until use. Unless otherwise stated, 50 oocytes or embryos per lane were used. After blocking with 5% skim milk (BD Biosciences) in TBST (0.1% Tween 20) for 1 h, the membranes were washed with TBST and probed with anti-phospho Stat3 (Tyr705) (1:1000, #9145, CST), anti-Stat3 (1:1000, #12640, CST), or anti-α-Tublin antibody (1:2000, #2125, CST) with 5% Blocking One-P (Nacalai Tesque) in TBST at 4 °C for overnight. The membranes were washed with TBST and incubated with HRP-conjugated anti-rabbit IgG secondary antibody (1:2000, #7074, CST) in blocking buffer at room temperature for 1 h. The membranes were washed with TBST and processed using the Chemi-Lumi One Ultra (Nacalai Tesque). Immunoblots were visualized using either Hyperfilm ECL (GE Healthcare) or ImageQuant LAS 500 (GE Healthcare). Quantification was performed using ImageQuant TL8.1 (GE Healthcare, Chicago, IL, USA).
4
Western Blot Analysis of ALDH7A1
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The control siRNA or cells were lysed with RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 0.25% Na deoxycholate, 5 mM EDTA, 1% Triton X-100, 5 mM EGTA, and 1% protease inhibitor cocktail) on ice for 30 minutes. The lysates were centrifuged at 14,000 rpm for 20 minutes. Equal amounts of protein samples were separated by SDS-PAGE and were then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking in 5% of BSA or milk, the membrane was incubated with anti-ALDH7A1 or actin antibodies. Blots were rinsed three to four times with PBS and incubated with horseradish peroxidase-conjugated secondary antibody. The blots were exposed to ECL Plus detection reagent. The images were analyzed by using Image QUANT TL8.1 (GE Healthcare, Marlborough, MA, US).
5
Western Blotting Analysis of Signaling Pathways
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Total cellular extracts and western blotting were performed as previously described [40 (link), 41 (link)] using antibodies against phospho-Erk1/2 (Thr202/Tyr204) (clone 20G11, 1/500), Erk1/2 (clone I37F5, 1/500), NF-κB Phospho-p65 (Ser536) (clone 93H1, 1/500), NF-κB p65 (clone E498, 1/500), phospho-SAPK/JNK (Thr183/Tyr185) (9251, 1/500), SAPK/JNK (clone 56G8, 1/500), p53 (9282, 1/500), phospho-p53 (Ser15) (9284, 1/500), from Cell Signaling Technology (Ozyme, Saint Quentin Yvelines, France), hCNT1 (clone H-70, 1/200), NF-κB p50 (H-119, 1/500), MRP2 (H-17, sc-5770, 1/500), Bax (N-20, 1/500), BclXL (H-5, 1/500) from Santa Cruz Biotechnology Inc. (Heidelberg, Germany) or hCNT3 (HPA023311; 1/500), β-actin (A5441, 1/5000) from Sigma-Aldrich (St. Quentin Fallavier, France). Antibodies were diluted in 5% (w/v) non-fat dry milk in Tris-Buffered Saline Tween-20 (TBS-T). Peroxydase-conjugated secondary antibodies (Sigma-Aldrich) were used and immunoreactive bands were visualised using the West Pico chemoluminescent substrate (Thermo Scientific, Pierce, Brebières, France). Chemo-luminescence was visualised using LAS4000 apparatus (Fujifilm). Densities of bands were integrated using Image Quant TL 8.1 (GE Healthcare Life Sciences, Velizy-Villacoublay, France) and represented as histograms. Three independent experiments were performed.
6
Western and Southern Blotting Protocols for HAE Cell Analysis
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For Western blotting, the HAE cells on the insert of the ALI culture were lysed in 200 μl of 1 × SDS-loading buffer. Lysed samples were loaded for SDS-polyacrylamide gel electrophoresis (PAGE), transferred, and blotted with antibodies as indicated in the figures, as previously described [16 (link)]. Images were developed under the imager FUJIFILM LAS 4000 (FUJIFILM Life Sciences) and quantified with Multi Gauge V2.3 software (FUJIFILM Life Sciences).
For Southern blotting, HAE cells were trypsinized off the insert of the ALI culture, washed and collected for extraction of low molecular weight (Hirt) DNA [73 (link),74 (link)]. Southern blotting was performed using an HBoV1 NS and Cap gene probe, as previously described [27 (link)]. A mitochondrial DNA probe was used as a control for the recovery of the Hirt DNA [75 (link)]. Images were developed with a Typhoon FLA 9000 phosphor imager and quantified using ImageQuant TL 8.1 (GE Healthcare).
For Southern blotting, HAE cells were trypsinized off the insert of the ALI culture, washed and collected for extraction of low molecular weight (Hirt) DNA [73 (link),74 (link)]. Southern blotting was performed using an HBoV1 NS and Cap gene probe, as previously described [27 (link)]. A mitochondrial DNA probe was used as a control for the recovery of the Hirt DNA [75 (link)]. Images were developed with a Typhoon FLA 9000 phosphor imager and quantified using ImageQuant TL 8.1 (GE Healthcare).
7
Quantitative RNA and Protein Analysis
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Total RNA was extracted using Trizol (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA synthesis and qPCRs were performed as described (Grönroos et al., 2012 (link)). Primer sequences are listed in Supplementary file 3 . Total cell lysates were generated as described (Germain et al., 2000 (link)), and Western blotted using the antibodies listed in Supplementary file 3 . Western blots were imaged and quantified using an ImageQuant 4000 mini and ImageQuant TL 8.1 software (GE Healthcare, UK).
8
In Vitro Translation of TSWV RNAs
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TSWV gRNA and sgRNA constructs were linearized with Sma and served as the templates for RNA transcription using T7 RNA polymerase. The 20 μL in vitro translation mixture contained 10 μL WGE (Promega), 0.5 pmol RNA template, 0.8 μL 1 mM amino acids mix (without methionine), 100 mM potassium acetate, and 0.5 μL [5 μCi] 35S-methionine. The translation mixture was incubated at 25 °C for 1 h and then resolved on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The gel was dried and subjected to Fujifilm Phosphorimager screening for 3 h. The screen was subsequently scanned by an Amersham Typhoon fluorescent image analyzer. Radioactive band intensity was quantified using ImageQuant TL 8.1 (GE Lifesciences, Piscataway, NJ, USA). All experiments were repeated at least three times.
9
Western Blot Analysis of Adipose Tissue
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Protein lysates were generated from ≈80 mg of finely cut adipose tissue by homogenization in lysis buffer 28 using a tissue lyzer (Qiagen, Germany). Immediately after homogenization, SDS was added to a concentration of 2%. Samples were then centrifuged at 16,000g for 20 min at 4°C to generate lysates. The bicinchoninic acid assay (Thermo Fischer Scientific, MA) was used to determine the protein concentration in the samples and samples were adjusted to a protein concentration of 0.5 µg/µl in sample buffer.
An equal amount of protein was separated by SDS‐polyacrylamide gel electrophoresis (SDS PAGE) and blotted on to a PVDF membrane by semidry blotting. The membrane was then blocked for 1 h in 3% fish gel (FG) (Sigma Aldrich, Copenhagen, Denmark) and incubated overnight in primary antibody against AMPKα1 protein (Hardie G.), AMPKThr172 phosphorylation (#2535s Cell Signaling Technologies (CST), Danvers, MA), HSL protein, and HSLSer660 and Ser565 as well as perilipin (#8334S CST), GLUT4 protein (#PAI‐1065, ABR), PEPCK protein (#10004943, Cayman Chemical Co., Ann Arbor, MI), PDK4 protein (Hardie G.), PDH‐E1α protein (Hardie G.), PDHSer300 phosphorylation (Hardie G.), and PDHSer232 phosphorylation (#AP1063, Millipore). The following day the membrane was incubated in secondary antibodies (Dako) in 3% FG and quantified using LAS 4000 and Image Quant TL 8.1 (GE Healthcare, Germany).
An equal amount of protein was separated by SDS‐polyacrylamide gel electrophoresis (SDS PAGE) and blotted on to a PVDF membrane by semidry blotting. The membrane was then blocked for 1 h in 3% fish gel (FG) (Sigma Aldrich, Copenhagen, Denmark) and incubated overnight in primary antibody against AMPKα1 protein (Hardie G.), AMPKThr172 phosphorylation (#2535s Cell Signaling Technologies (CST), Danvers, MA), HSL protein, and HSLSer660 and Ser565 as well as perilipin (#8334S CST), GLUT4 protein (#PAI‐1065, ABR), PEPCK protein (#10004943, Cayman Chemical Co., Ann Arbor, MI), PDK4 protein (Hardie G.), PDH‐E1α protein (Hardie G.), PDHSer300 phosphorylation (Hardie G.), and PDHSer232 phosphorylation (#AP1063, Millipore). The following day the membrane was incubated in secondary antibodies (Dako) in 3% FG and quantified using LAS 4000 and Image Quant TL 8.1 (GE Healthcare, Germany).
10
Measuring Radioactivity in Deciduous Teeth
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We measured radioactivity in deciduous teeth using BAS-MS 2040 IP (FUJIFILM Corp., Tokyo, Japan). The teeth were positioned such that their labial/buccal surfaces faced the IP surface, then IP cassettes were placed in a shielding box made of lead with iron plates for 4 weeks to block natural background radiation (Figure S2). Details of how the teeth were positioned on the IP are described in the SI. The amount of radioactivity in each tooth was determined as QLs using a Fuji FLA-7000 bio-imaging analyser (FUJIFILM Corp.) and ImageQuant TL 8.1 (GE Healthcare). We used nine IPs to measure radioactivity in thousands of samples. However, the sensitivity of the IP varied, and the QLs differed even when the same sample was measured using different IP. Therefore, we normalised the QLs obtained from different IPs by placing a reference scale of potassium chloride on each one to calibrate the radioactivity values (Figure S2). The methods used to normalise the QLs are detailed in the SI.
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