Cell lysis, protein extraction, and immunoblotting were performed as described83 (link). Protein concentrations were quantified using Bradford assays (Bio-Rad) with bovine serum albumin (BSA) as standard, and a SpectraMax M5 microplate reader. Samples were resolved by SDS-PAGE on 4–12% gradient polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and detected by immunoblotting. As primary antibodies, we used rabbit monoclonal antibodies to Smad2, phospho-Smad2 (Ser465/467), Smad3, Akt, and phospho-Akt (p-AktS473) from Cell Signaling. Antibody against phospho-Smad3 (Ser423/425) was from Abcam, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Sigma. For immunoblotting, immunoreactive bands were visualized using Western Lighting Plus ECL (Perkin Elmer).
Western lighting plus ecl
Manufactured by PerkinElmer
Sourced in United States
The Western Lighting Plus-ECL is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It is designed to provide a high-intensity, stable signal for the visualization of target proteins.
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Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
Bradford assay, by Bio-Rad (4 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Immobilon p, by Merck Group (3 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Chemidoc touch, by Bio-Rad (2 mentions)
Complete edta free, by Roche (2 mentions)
Protein g sepharose bead, by GE Healthcare (2 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated donkey anti mouse secondary antibody, by Jackson ImmunoResearch (2 mentions)
Amersham hybond, by GE Healthcare (2 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (2 mentions)
Ibright fl1000 imaging system, by Thermo Fisher Scientific (2 mentions)
Biospectrum imaging system, by Analytik Jena (2 mentions)
Leupeptin, by Cell Signaling Technology (2 mentions)
Nupage 4 12 bistris mini protein gels, by Thermo Fisher Scientific (2 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ha, by Thermo Fisher Scientific (2 mentions)
55 protocols using western lighting plus ecl
1
Immunoblotting Analysis of Smad and Akt Signaling
2
Immunoprecipitation and Western Blot Analysis
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Whole cells were collected and lysed in ice-cold lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl pH 7.4, 0.5% Triton X-100 and protease inhibitor cocktail (Roche, Basel, Switzerland) and homogenized by sonication in 1.5-ml microfuge tubes on ice for 30 s. Protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA). Cellular extracts (200 μg) were incubated with the indicated antibody overnight, followed by a 1-h incubation with Protein A/G beads or antibody-conjugated beads overnight at 4 °C. The immunocomplexes were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were then incubated with the indicated primary antibody followed by an HRP-conjugated secondary antibody. Immunoreactive bands were detected using Western Lighting Plus-ECL (PerkinElmer, Waltham, MA).
3
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA buffer (20 mM Tris, pH 7.3, 150 mM NaCl, 5 mM EDTA, 25 mM NaF, 25 mM sodium pyrophosphate, 1% TritonX, 0.1% SDS, 0.5% deoxycholate, 10% glycerol), sonicated, and centrifuged at 14,000g at 4 °C for 10 min. Protein from aortic tissue was extracted using TRIzol™ Reagent (ThermoFisher) according to Kopec et al.’s28 (link) protocol. Protein concentrations were determined by DC™ protein assay (Bio-Rad). The protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were then incubated with primary antibodies at 4 °C overnight and subsequently with secondary antibodies at room temperature for an hour. Protein bands were detected by Western Lighting Plus-ECL (PerkinElmer) using X-ray films. Antibodies are listed in Supplemental Table 2 .
4
Western Blot Analysis of EMT Markers
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Cells were washed with PBS, and proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma) and phosphatase inhibitor cocktail 2 (Sigma). The concentration of extracted protein was measured by Bradford protein assay (Bio‐Rad, Hercules, CA, USA). Electrophoresis was performed on 7.5% acrylamide gels, and proteins were transferred onto polyvinylidenefluoride (PVDF) membranes (Bio‐Rad). Primary antibodies against PODXL, GAPDH, vimentin, E‐cadherin, N‐cadherin, Fibronectin, αSMA, Smad2, phosphor‐Smad2, Akt, phospho‐Akt, SnaI and twist were diluted at 1:250, 1:2000, 1:500, 1:500, 1:200, 1:500, 1:100 1:1000, 1:1000, 1:1000, 1:1000, 1:200 and 1:200, respectively. Secondary antibodies (anti‐mouse IgG HRP‐linked antibody and anti‐rabbit IgG, HRP‐linked antibody [Cell Signaling Technology]) were diluted at 1:2000. Cell extracts were detected using the Western Lighting Plus‐ECL (PerkinElmer, Boston, MA, USA). The figures were photographed using Chemi Doc Touch (Bio‐Rad). These results were attained from more than three independently performed experiments.
5
HeLa Cell Transfection and Immunoprecipitation
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HeLa cells were cultured on 100-mm dishes or multiwell plates in DMEM (Quality Biological) supplemented with 10% fetal bovine serum (Corning), 100 units/ml penicillin, and 100 μg/ml streptomycin (Corning) at 37°C under a humidified atmosphere (95:5 air:CO2). Cells plated on 100-mm dishes or 6-well plates were transfected with 7 μg or 1.4 μg of plasmid DNA, respectively, using the X-tremeGENE9 DNA transfection reagent (Roche) as described previously (Mattera et al., 2020b (link)). Cells were washed twice with PBS and lysed in 50 mM Tris-HCl pH 7.4, 0.8% (vol/vol) Triton X-100 and 75 mM NaCl supplemented with a protease inhibitor cocktail (EDTA-free Complete, Roche). Lysates from cells cultured on 100-mm dishes were subjected to immunoprecipitation using 2 μg of antibodies immobilized onto 25 μl of Protein G-Sepharose beads (GE Healthcare) as described previously (Mattera et al., 2003 (link)). Lysates or immunoprecipitated complexes were subjected to SDS–PAGE in 10% acrylamide gels and transferred onto nylon membranes (Immobilon-P, Millipore). Membranes were subsequently stained with Ponceau S (Sigma Aldrich) and immunoblotted with primary and secondary antibodies using buffers containing 0.05% Tween (Sigma Aldrich) and 3% dry milk (BioRad). Blots were developed with Western Lighting Plus-ECL (PerkinElmer) or SuperSignal West Pico Plus (ThermoFisher) reagents.
6
Western Blot Analysis of Osteocalcin
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Cells were homogenized directly into the following buffer: Tris 50 mM, NaCl 150 mM, EDTA 10 mM, and Triton-X 1% and centrifuged at 10,000g for 10 min. Protein concentrations were determined by the Bradford assay. Proteins were resolved by 16% SDS-PAGE, electrotransferred on PVDF membranes (Amersham™ Hybond™, GE Healthcare Life Science, cat. number 28906837), and blocked with 5% [v/v] nonfat dry milk/0.1% [v/v] TBS-T. The blots were probed with the following primary antibodies: mouse monoclonal anti-osteocalcin (1 : 500) (abcam-ab13420) in 5% BSA/TBS-T 0.1% and mouse monoclonal anti-beta actin (1 : 10,000) (sigma-aldrich-A5541).
Membranes were then incubated with the appropriate horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (1 : 5000; Jackson), and the reaction was detected with the Western lighting Plus ECL (Perkin Elmer, Waltham, MA, USA).
Membranes were then incubated with the appropriate horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (1 : 5000; Jackson), and the reaction was detected with the Western lighting Plus ECL (Perkin Elmer, Waltham, MA, USA).
7
Western Blot Protein Detection Protocol
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Cells were lysed in RIPA buffer (VWR International) containing freshly added protease inhibitor complex (Roche). 20 μg of protein was loaded on precast NuPAGE Bis-Tris gels (Thermo Fisher Scientific) followed by transfer onto nitrocellulose. Chemiluminescent signals were detected with Western Lighting Plus-ECL (Perkin Elmer, NEL103001) and imaged with the ChemiDoc Touch Imaging System (Bio-Rad). Anti-PHGDH antibody was purchased from Sigma (WH0026227M1), and was used at 1:1000 dilution. Anti-β-ACTIN antibody was purchased from Cell Signaling (3700S), and was used at 1:2000 dilution. Uncropped western blot images are reported in Supplementary Figure 13 .
8
Protein Immunoblotting of MQ-Treated Cells
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B16 cells or RAW cells were plated in 10- to 15-cm tissue culture dishes in complete RPMI media. Twenty-four hours later, they were treated with MQ (concentration as indicated) in RPMI media devoid of nonessential amino acids for 4 hours. The cells were harvested 24 hours after treatment and total protein lysates were prepared using RIPA buffer (Cell Signaling Technology, catalog 9806S) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein lysates were separated on precast 4%–12% polyacrylamide NuPAGE Novex Bis-Tris gels (Invitrogen) and then electrotransferred onto PVDF membranes (EMD Millipore). The membranes were blocked and incubated with the respective primary and secondary antibodies. The antibodies used are listed in the Supplemental Methods . Chemiluminescence was detected with the Western Lighting Plus ECL (PerkinElmer) or SuperSignal West Pico reagent (Thermo Fisher Scientific/Pierce) using the Invitrogen iBright FL1000 imaging system.
9
Western Blot Analysis of Cardiac Proteins
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Total soluble protein was extracted from the whole heart tissues (following 30 min ischemia and 1 hr reperfusion or 24 hr reperfusion) with extraction buffer. Seventy-five (75 μg) proteins from each sample were separated by SDS-PAGE and transferred onto nitrocellulose membrane (0.2 um pore size). The membrane was incubated in cold conditions overnight with primary antibodies (phospho-serine473-AKT, AKT, phospho-serine235/236-S6, S6, p-ERK1/2, ERK1/2, p-P38, P38, phospho-tyrosin705-STAT3, STAT3, Bcl-2, Bax, SOD-2, and ferritin heavy chain 1 or GAPDH-HRP) in a 1 : 1000 dilution with 5% BSA. The membrane was washed and incubated with horseradish peroxidase conjugated secondary antibody (1 : 3000 dilution in 5% milk solution) and the blots were developed using a chemiluminescent system (Western Lighting Plus-ECL; Perkin Elmer, Inc.).
10
Western Blot Analysis of SVCT2, RIPK1, and MLKL
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N2a and HN33.11 cells were lysed with NP-40 buffer supplemented with a protease/phosphatase inhibitor cocktail (Cell Signaling #5872). The proteins (30–50 μg) were separated using 10% SDS-PAGE gel, transferred to PVDF membranes (0.45-μm pore; Immobilon-P #IPVH00010, Merck Millipore) and probed with anti-SVCT2 (1:1000), anti-RIPK1 (1:3000) and anti-MLKL (1:5000) overnight. The membranes were then incubated with secondary antibodies, including HRP-conjugated donkey anti-goat IgG, donkey anti-rabbit IgG, donkey anti-rat IgG (1:6000) and anti-actin-HRP (1:50000) for 2 h at room temperature. The reaction was developed using the Western Lighting® Plus-ECL enhanced chemiluminescence substrate (PerkinElmer, Waltham, MA, USA).
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