Washed platelets (4 × 108/mL) were stimulated as indicated and lysed directly in 4X NuPAGE sample buffer containing 0.5 M dithiothreitol (DTT). Proteins were resolved by electrophoresis as previously described45 (link) and transferred onto polyvinylidene fluoride (PVDF) membranes. For some experiments, gels and membranes were cut into two parts to be probed separately with different primary antibodies47 (link). Proteins were visualized by near-infrared detection using a LI-COR® Odyssey imaging system (providing a wide linear dynamic range) unless indicated otherwise. LI-COR® Image Studio (LI-COR®, Cambridge, UK) was used to create final images, the software analysis options only affect how raw data pixels are mapped to the screen and does not alter the experimental data. LI-COR® Image Studio (LI-COR®, Cambridge, UK) was used to quantify bands. Bands were defined using the rectangle shape tool to obtain fluorescence values and median local background (intensity of pixels in a border around the shape) was automatically subtracted.
Image studio
Manufactured by LI COR
Sourced in United States, United Kingdom, Germany
Image Studio is a software application that enables the analysis and quantification of data from a variety of imaging platforms, including those produced by LI-COR. The software provides tools for visualizing, processing, and interpreting image data, facilitating the scientific workflow.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx, by LI COR (50 mentions)
Odyssey blocking buffer, by LI COR (38 mentions)
Odyssey imaging system, by LI COR (17 mentions)
Trans blot turbo transfer system, by Bio-Rad (17 mentions)
Odyssey fc imaging system, by LI COR (17 mentions)
Ripa buffer, by Thermo Fisher Scientific (17 mentions)
Odyssey clx infrared imaging system, by LI COR (16 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Odyssey clx imager, by LI COR (14 mentions)
Polyacrylamide gel, by Thermo Fisher Scientific (14 mentions)
Pvdf membrane, by Merck Group (13 mentions)
Revert total protein stain, by LI COR (13 mentions)
Immobilon membrane, by Merck Group (13 mentions)
Protease inhibitor cocktail, by Roche (11 mentions)
Odyssey clx imaging system, by LI COR (10 mentions)
Odyssey clx system, by LI COR (10 mentions)
Odyssey infrared imaging system, by LI COR (10 mentions)
Odyssey imager, by LI COR (9 mentions)
Odyssey scanner, by LI COR (9 mentions)
Protease inhibitor cocktail, by Merck Group (8 mentions)
447 protocols using image studio
1
Immunoblotting Quantification of Washed Platelets
2
Fluorescence Intensity Quantification in Organs
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Equally sized ROIs were manually drawn using the software Image Studio (version 5.2, Licor, Lincoln, NE, USA) from Li-Cor. Examples of ROIs are shown in Figure 1 . Brain and right kidney ROIs were drawn from the prone view images while liver and front left paw ROIs were drawn from the supine view images. The average fluorescence intensity of each ROI was calculated and generated by Image Studio (version 5.2, Licor, Lincoln, NE, USA).
3
Generation of Stable Cell Lines
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To establish polyclonal knockdown or overexpression lines, EAhy926 cells (cultured in DMEM containing 10% FBS, 1% sodium pyruvate, 1% penicillin/streptomycin, and 2% HAT supplement; Gibco) were incubated with the viral supernatant and 8 µg/ml polybrene for 18 h, and infected cells were selected with 2 µg/ml Puromycin (pLKO) or 50 µg/ml Hygromycin (CMV-pLENTI-Hygro) as was appropriate. Knockdown was assessed by immunoblotting of stable lines lysed in RIPA buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor mixture tablets (Roche). Immunoblotting was performed with fluorescent secondary antibodies (IR Dye800 or IR Dye680; LI-COR Biosciences) and scanned on the Odyssey CLx infrared imaging system. Quantification was performed on the raw images in Image Studio (LI-COR Biosciences); lanes were defined and bands were automatically identified. The profile feature was used to identify band boundaries and lane background was subtracted. The signal for the protein of interest was normalized to that of the loading control. Optimization of images for publication was performed by adjusting the brightness and contrast in Image Studio (LI-COR Biosciences).
4
Immunoblotting Analysis of Glutamate Receptor Subunits in Brain Regions
5
Cell Lysis and Western Blot Analysis
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Cells were lysed in IP-MS buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 [NP-40] [vol/vol], 0.5% sodium deoxycholate [wt/vol], 10 mM b-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM NaF, 2 mM Na3VO4, and cOmplete protease inhibitor cocktail tablets [MilliporeSigma]). Samples were cleared by centrifugation and protein concentration determined via the BCA assay (Thermo Fisher Scientific). 20–30 μg of protein were loaded in SDS–PAGE gels and transferred to PVDF or Nitrocellulose membranes. These were then blocked with TBS-Tween 20 (TBS-T) 5% nonfat milk (wt/vol) and incubated with primary antibodies. HRP or infrared dye-conjugated secondary antibodies were used for electrochemiluminescence or infrared detection using a ChemiDoc MP (Bio-Rad) or an Odyssey instrument (LI-COR). Figures were assembled using Image Lab (Bio-Rad), Image Studio (LI-COR), Inkscape 1.2 (Inkscape), and Illustrator (Adobe). Densitometric analyses were performed using ImageJ (NIH) or Image Studio (LI-COR) and data were analyzed in Excel (Microsoft) and GraphPad Prism 9 (GraphPad).
6
Phospholipid Quantification by Densitometry
7
Western Blot Analysis of GABAA Receptor
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Samples were homogenized in HEPES-buffered sucrose (320 mM sucrose, 4 mM HEPES, pH 7.4, 1% SDS), separated by electrophoresis on Novex 4–12% Tris-Glycine gels (Invitrogen) and transferred to PVDF membrane (Millipore). Transfer was performed at 4°C in a buffer containing 35 mM Tris, 192 mM glycine, and 20% methanol. Membranes were blocked using 5% milk and later incubated with the primary antibody, anti-GABAA receptor delta subunit (1:1000, Novus). Protein levels were normalized with GAPDH (1:5000 Millipore). HRP-conjugated secondary anti-mouse or anti-rabbit antibodies (1:10000, GE Healthcare) were used. Membranes were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and densitometric analysis was performed with Image StudioTM (LI-COR Bioscience).
8
Histone H3 Acetylation Analysis
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Frontal cortex of vehicle, imidazenil and diazepam‐treated rats were homogenized in HEPES‐buffered sucrose (320 mmol L−1 Sucrose, 4 mmol L−1 HEPES pH 7.4, 1% SDS). We used Pierce® Bicinchoninic acid (BCA) assay to determine protein concentration (Thermo Fisher Scientific, Waltham, MA). Lysates were resuspended in Laemmli reducing buffer and 30 μg of each sample were separated by electrophoresis on Novex 4%‐12% Tris‐Glycine gels. Proteins were then transferred to PVDF membrane in a buffer containing 35 mmol L−1 Tris, 192 mmol L−1 glycine, and 20% methanol at 4°C. Membranes were blocked using 5% milk and later incubated with the primary antibody, anti‐H3 (1:1000) or anti‐acetyl‐H3 (1:1500). Protein levels were normalized with GAPDH (1:5000). HRP‐conjugated secondary anti‐mouse or anti‐rabbit antibodies (1:10 000) were used. Membranes were developed with Immobilon Western Chemiluminescent HRP Substrate and densitometric analysis performed with Image StudioTM (LI‐COR, Bioscience). The expression levels for the respective proteins were normalized to GAPDH protein levels.
9
Whole Cell and Chromatin Protein Extraction
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For whole cell extracts, cells were harvested by trypsinisation, washed twice in ice-cold PBS, pelleted by centrifugation, and boiled in boiling in 1x SDS loading buffer. Chromatin extracts were prepared as described previously31 (link). All protein extracts were resolved by SDS-PAGE, transferred on to PVDF membranes (Millipore, 0.45 µM pores) and blocked with 5% milk/TBS-T. Membranes were probed with primary antibodies overnight, washed extensively and incubated with relevant HRP-conjugated secondary antibodies for 1 h (IRDye 800 CW-or IRDye 700 CW-Secondary (1Li-cor)). After further washing, luminescence signal generated using Immobilon Western Chemiluminescent HRP substrate (Millipore) was detected using autoradiography or the Odyssey® XF Imaging system (LI-COR Biosciences). LI-COR images were quantified with Image StudioTM. Antibodies are listed in Supplementary Data 2 .
10
Heregulin-Induced Signaling in Tumor Cells
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Tumour cells were grown to near confluency in 6-well culture plates containing 5 ml of 10% FBS growth medium. Cells were washed once with 5 ml of 0.5% FCS/medium and then incubated in 5 ml of fresh 0.5% medium containing no drug (control), a TKI, or a cytotoxic drug for 24 h at 37°C, prior to treatment with the HER ligand heregulin (20 nM) (R&D Systems) for 15 min at 37°C. Tumour cells were then lysed in 400 μl of lysis buffer (Invitrogen, Paisley, UK) containing the cocktail protease inhibitor (Sigma-Aldrich). Cell lysates were heated to 75°C for 10 min, then 30 μl of protein samples (30 μg) were separated on 4–12% Bis-Tris gels (Invitrogen) and transferred onto polyvinylidene difluoride (PVDF) membranes using the XCell II Mini-Cell Blot Module kit (Invitrogen). PVDF membranes were probed with antibodies using SNAP i.d systems (Millipore, Watford, UK). Signal for all were detected using either the Western Breeze chemiluminescence kit (alkaline-phosphatase conjugated secondary antibody, Invitrogen) and visualized by the G-box imaging system (Syngene, Cambridge UK) or a fluorescence conjugated secondary antibody (LI-COR Ltd) and visualized by the accompanying software, LI-COR Image Studio.
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