Pulmonary arteries collected from 22 rats were used in Western blot analysis, one sample was missing due to technical issues. Total protein content in pulmonary arteries was quantified using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Massachusetts, USA). Samples were measured at 562 nm (PHERAstar, BMG Labtech, Ortenberg, Germany) after 30 min incubation at 37 °C. The supernatant was used to prepare gels while in Laemmli sample buffer containing 2% SDS. 12% Criterion Precast Gel (Bio-Rad laboratories, Copenhagen, Denmark) was used to separate proteins, which were then electrotransferred to a nitrocellulose membrane. Blots were blocked with 5% nonfat dry milk in PBS-T as described previously49 (link). After washing with PBS-T, blots were incubated with primary antibodies (COX-1, COX-2 and beta-actin) overnight at 4 °C, and visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature using the enhanced chemiluminescence system (ECL Plus, GE Healthcare, Amersham, United Kingdom). Values were normalized to total protein using Stain-Free technology.
Criterion precast gel
Manufactured by Bio-Rad
Sourced in United States, Italy
Criterion precast gels are electrophoresis gels used for the separation and analysis of proteins. They are pre-cast and ready-to-use, providing a convenient and consistent solution for protein electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (6 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Imagequant las 4000 mini, by GE Healthcare (4 mentions)
Enhanced chemiluminescence, by Cytiva (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Immobilon, by Merck Group (3 mentions)
Tsg101, by GeneTex (3 mentions)
Wbkls0500, by Merck Group (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Protein assay, by Bio-Rad (3 mentions)
Anti β actin antibody, by Merck Group (3 mentions)
Polyacrylamide gel, by Bio-Rad (3 mentions)
Odyssey quantitative fluorescence imaging system, by LI COR (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Trans blot turbo system, by Bio-Rad (3 mentions)
Chemiluminescent detection reagents, by Bio-Rad (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl plus, by GE Healthcare (2 mentions)
71 protocols using criterion precast gel
1
Quantifying Pulmonary Artery Protein Expression
2
Protein Separation and Visualization
3
Western Blot Protein Analysis
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Cells were lysed with Laemmli buffer including phosphatase and protease inhibitors (Thermo Scientific, 78420, 1862209). Twenty micrograms of proteins were separated on 4–15% Criterion precast gel (567-1084, Bio-Rad) and transferred on nitrocellulose membrane (Biorad). Western blottings were developed with chemiluminescence horseradish peroxidase substrate (Millipore, WBKLS0500) on a Luminescent image Analyser, ImageQuant LAS 4000 mini (GE Healthcare). Bands were quantified using ImageJ. See Supplementary Fig. 5 for the uncropped immunoblots.
4
Two-Dimensional Gel Electrophoresis of Proteins
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2D gel electrophoresis was performed as described by Jameson-Lee et al.41 (link) Briefly, eluted samples were boiled with SDS-PAGE loading buffer and resolved on Bio-Rad Criterion Precast gel (12 well, 4–12% acrylamide) under nonreducing conditions. The lanes were excised and treated in warm 100 mM DTT for 15 min. Free sulfhydryls were alkylated by 100 mM iodoacetamide in Laemmli loading buffer without bromophenol blue for 5 min. The gel strip was then placed in a 1-well Criterion Precast gel (6–16% acrylamide). The strip was locked in place by addition of an agarose overlay (2-D starter Kit, Bio-Rad) before gel electrophoresis. Proteins were visualized with silver stain.
5
Quantitative Western Blot Analysis
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For cellular protein lysates, cells were scraped on ice using cold Ripa lysis buffer (150 nM NaCl, 50 mM Tris–HCl pH 8, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (CompleteTM, Roche), 1 mM Na3VO4 (Sigma), 100 mM NaF (Sigma), and 1 mM DTT (Sigma).
Proteins were separated in 4–20% SDS–PAGE (Criterion Precast Gel, Bio‐Rad) and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% dried milk in TBS‐0,1% Tween 20 or in Odyssey Blocking Buffer (LI‐COR, Biosciences) and incubated at 4°C overnight with primary antibodies. The list of primary antibodies is provided in Appendix TableS5 .
Membranes were washed in TBS‐0,1% Tween 20 and incubated 1 h at RT with IR‐conjugated (AlexaFluor680, Invitrogen or IRDye 800, Rockland) secondary antibodies for infrared detection (Odyssey Infrared Detection System, LI‐COR) or with the appropriate horseradish peroxidase‐conjugated secondary antibodies (GE Healthcare) for ECL detection (Clarity Western ECL Substrate, Bio‐Rad). Band quantification was performed using the Odyssey v1.2 software (LI‐COR) or the QuantiONE software (Bio‐Rad Laboratories). The Re‐Blot Plus Strong Solution (Millipore) was used to strip the membranes, when reblotting was needed.
Proteins were separated in 4–20% SDS–PAGE (Criterion Precast Gel, Bio‐Rad) and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% dried milk in TBS‐0,1% Tween 20 or in Odyssey Blocking Buffer (LI‐COR, Biosciences) and incubated at 4°C overnight with primary antibodies. The list of primary antibodies is provided in Appendix Table
Membranes were washed in TBS‐0,1% Tween 20 and incubated 1 h at RT with IR‐conjugated (AlexaFluor680, Invitrogen or IRDye 800, Rockland) secondary antibodies for infrared detection (Odyssey Infrared Detection System, LI‐COR) or with the appropriate horseradish peroxidase‐conjugated secondary antibodies (GE Healthcare) for ECL detection (Clarity Western ECL Substrate, Bio‐Rad). Band quantification was performed using the Odyssey v1.2 software (LI‐COR) or the QuantiONE software (Bio‐Rad Laboratories). The Re‐Blot Plus Strong Solution (Millipore) was used to strip the membranes, when reblotting was needed.
6
Ubiquitin Protein Detection by SDS-PAGE and Western Blot
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All fractions were subjected to one-dimensional
gel electrophoresis on an 8–16% Criterion precast gel (Bio-Rad)
at 200 V, 50 mA, and 15 W for 56 min, followed by transfer to a PVDF
membrane (EMD Millipore, Billerica, MA) at 100 V, 350 mA, and 35W
for 1 h. Free ubiquitin, polyubiquitin, and ubiquitinated proteins
were detected by blotting with anti-ubiquitin antibody 3933 (Cell
Signaling Technology) followed by anti-mouse IgG-HRP (Cell Signaling
Technology). Protein bands were visualized with an Image Lab System
(Bio-Rad, Hercules, CA) using the Gel-Doc program (Kodak Molecular
Imaging Systems) and the SuperSignal West Dura chemiluminescent substrate
(Thermo Fisher Scientific, Waltham, MA).
gel electrophoresis on an 8–16% Criterion precast gel (Bio-Rad)
at 200 V, 50 mA, and 15 W for 56 min, followed by transfer to a PVDF
membrane (EMD Millipore, Billerica, MA) at 100 V, 350 mA, and 35W
for 1 h. Free ubiquitin, polyubiquitin, and ubiquitinated proteins
were detected by blotting with anti-ubiquitin antibody 3933 (Cell
Signaling Technology) followed by anti-mouse IgG-HRP (Cell Signaling
Technology). Protein bands were visualized with an Image Lab System
(Bio-Rad, Hercules, CA) using the Gel-Doc program (Kodak Molecular
Imaging Systems) and the SuperSignal West Dura chemiluminescent substrate
(Thermo Fisher Scientific, Waltham, MA).
7
EMILIN1 Detection in Lymphedema
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Mice were sacrificed at day 4 or 7 after surgery, and lymph samples were collected from tails and incubated with recombinant EMILIN1 for 18 h. Whole lysates of both human samples and tail tissues from normal and lymphoedematous sites were prepared using thiourea/urea lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS). Samples were subjected to SDS/4–12%PAGE (using Criterion Precast Gel, BioRad) and blotted on nitrocellulose membranes (Amersham Hybond-ECL, Amersham Pharmacia Biotech). Membranes were blocked (5% non-fat milk, 0.1% Tween-20 in TBS) and incubated with rabbit polyclonal anti-human EMILIN1 (As556) to identify recombinant human EMILIN1 protein or rabbit anti-mouse EMILIN1 (mC1q, an antibody generated to specifically recognize the mouse gC1q domain) to detect EMILIN1 in mouse tissue lysates. Horseradish peroxidase (HRP)-tagged secondary antibodies (Jackson Immunoresearch) were used at proper dilutions. Signals were detected using ECL reagents (Amersham Western Blotting Detection System and HyperFilm ECL, Amersham Pharmacia Biotech).
8
Adipose Tissue Protein Analysis
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Mouse adipose tissue homogenate (50 μg protein extracted by RIPA solution) was subjected to electrophoresis on 4–16% gradient polyacrylamide gels (Criterion precast gel; Bio‐Rad Laboratories Inc., Hercules, CA), transferred to polyvinylidene fluoride membranes, incubated in blocking buffer (5% non‐fat dry milk in TBS) and then incubated with primary antibody UCP1 (Abcam, Cambridge, MA) (1:500 dilution) or Tyrosine Hydroxylase (Cell Signaling, Beverly, MA) (1:1000 dilution), washed with TBST, and incubated with fluorescence‐conjugated secondary antibody (1:5000 dilution). The blots were developed with a Li‐COR Imager System (Li‐COR Biosciences, Lincoln, NE) and normalized with the control α‐Tubulin (Cell Signaling, Beverly, MA) (1:500 dilution).
9
Western Blot Analysis of Extracellular Vesicles
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Brain homogenates (10 μg protein), fibroblast lysates (10 μg protein), and EVs proteins (15 μl of the lysate corresponding to 25% of the EVs lysate total volume), were separated by 4–20% tris-glycine gel electrophoresis (Criterion precast gel, Bio-Rad, Hercules, CA) and transferred onto PVDF membranes (Immobilon, EMD Millipore, Billerica, MA). Membranes were incubated with antibodies to CD63 (1:250, Cat# sc-15363, Santa Cruz Biotechnology, Dallas, TX), Alix (1:1000, Cat# ABC40, EMD Millipore), TSG101 (1:1000, Cat# 4A10, GeneTex, Irvine, CA), Flotillin-1 (1:1000, Cat# 610821, BD Biosciences, San Jose, CA), Flotillin-2 (1:1000, Cat# 610384, BD Biosciences), rab35 (1:1000, Cat# 9690, Cell Signaling Technology, Danvers, MA), and β-actin (1:2500, Cat# ab8226, Abcam, Cambridge, MA). The secondary antibodies used were HRP conjugated anti-rabbit, and anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). Protein bands were quantified using ImageJ software (NIH, Bethesda, MD). All data are shown as the trisomic to diploid ratio for Ts2 and 2N mice, or for DS patients and 2N control subjects.
10
Western Blot Analysis of Phospho-GSK3β
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Whole cell extracts were prepared using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) with protease and phosphatase inhibitors (cOmplete - protease inhibitor cocktail tablets, and PhosSTOP - phosphatase inhibitor cocktail tablets; Roche), and total protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL). Cell lysates containing equal amounts of total protein (∼5–10 µg) were heated at 100°C for 5 minutes in laemmli buffer (Sigma-Aldrich), electrophoretically separated on a 10% SDS-polyacrylamide gels (Criterion Precast Gel; Bio-Rad, Hercules, CA), and transferred onto polyvinylidene difluoride (PVDF) membranes (Immun-Blot; Bio-Rad). Membranes were incubated with primary antibodies for phospho-GSK3β-Ser9 (p-GSK3β-S9; Cell Signaling Technology, Danvers, MA; 5558), GSK3β (t-GSK3β; Cell Signaling Technology; 9832) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam; ab8245). Appropriate horseradish peroxidase-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL) were used. Membranes were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and visualized using a Kodak Image Station 440CF.
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