The effects of silencing HIF-1α and HIF-1β expression on the expression of proteins related to cell proliferation and angiogenesis were assessed by immunoblot assays. Total proteins were extracted from HIF-1α- or HIF-1β-silenced HCC cells after 24 h of hypoxia induction. The proteins were separated according to their molecular weight via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride membrane (GE Healthcare/Amersham, Buckinghamshire, UK), and blotted with mouse monoclonal antibodies specific for the proteins of interest. The blots were developed using the enhanced chemiluminescence technique (PerkinElmer, Boston, MA, USA) according to the manufacturer’s instructions, and the level of expression of each protein was quantified and compared. For detection of secreted proteins, enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA) was performed according to the manufacturer’s instructions.
Enhanced chemiluminescence technique
Manufactured by PerkinElmer
Sourced in United States
Enhanced chemiluminescence (ECL) is a technique used for the detection and quantification of proteins in Western blot analysis. It involves the use of a chemiluminescent substrate that emits light when it reacts with the enzyme-labeled target protein. This technique provides a sensitive and reliable method for protein visualization and analysis.
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Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Mini protean tgx precast 7.5 gel, by Bio-Rad (2 mentions)
Bicinchoninic acid protein assay reagent, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by GE Healthcare (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Ebm 2 medium, by Lonza (1 mentions)
Protein a or g sepharose, by GE Healthcare (1 mentions)
Hybond c extra nitrocellulose membrane, by Cytiva (1 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (1 mentions)
Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ripa cell lysis buffer, by Cell Signaling Technology (1 mentions)
Hif 1α, by Fortis Life Sciences (1 mentions)
Mouse anti hif 1α, by Cell Signaling Technology (1 mentions)
7 protocols using enhanced chemiluminescence technique
1
Hypoxia Regulation of Proliferation and Angiogenesis
2
Immunoprecipitation and Western Blot Analysis
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ECs were lysed in a buffer containing 25 mM Tris–HCl, pH 7.6, 100 mM NaCl, 0.15% Tween-20, 5% glycerol, 0.5 mM ethylene glycol tetraacetic acid (EGTA) and Protease Inhibitor Cocktail (Sigma Aldrich), 2 mM phenylmethanesulfonylfluoride (PMSF), 2 mM MgCl2. Cell lysates were incubated for 20 min on wet ice, and then centrifuged at 15,000 g, 20 min, at 4 °C. The total protein amount was determined using the bicinchoninic acid (BCA) protein assay reagent (Life technologies). Equivalent amounts (800 μg) of protein were immunoprecipitated for 1 h with the antibody of interest, and immune complexes were recovered on Protein G-Sepharose (GE Healthcare). Immunoprecipitates were washed twice with lysis buffer, twice with the same buffer without Tween-20 and then separated by SDS–PAGE. Proteins were then transferred to a nitrocellulose membrane (Biorad, Hercules, CA, USA), probed with antibodies of interest and detected by an enhanced chemiluminescence technique (PerkinElmer, Waltham, MA, USA).
Uncropped scans of all blots are provided asSupplementary Figs 11–14 .
Uncropped scans of all blots are provided as
3
Endothelial Cell Surface Biotinylation
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Transduced ECs were starved for 3 h with EBM-2 medium (Lonza) at 37 °C, 5% CO2 in a humidified atmosphere. Cells were surface labeled with the cell impermeable HaloTag PEG-Biotin Ligand 3 µM at 4 °C for 15 min, stimulated or not with VEGF-A165 30 ng/ml for 10 min at 37 °C. ECs were lysed in buffer containing 25 mM Tris–HCl pH 7.6, 100 mM NaCl, 1% Triton X-100, 5% glycerol, 0.5 mM EGTA, 2 mM MgCl2, 1 mM PMSF, 1 mM Na3VO4 and protease inhibitor cocktail. Cellular lysates were incubated for 20 min on wet ice, and then centrifuged at 15,000 × g, 20 min, at 4 °C. The total protein amount was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce). Equivalent amounts (1 mg) of protein were immunoprecipitated for 2 h at 4 °C on streptavidin-conjugated beads. Immunoprecipitates were washed three times with lysis buffer with or without detergent and then separated by SDS–PAGE with Mini PROTEAN TGX precast 7.5% gel (BIO-RAD). Proteins were then transferred to a Trans-Blot Turbo™ PVDF Transfer (BIO-RAD), probed with antibodies of interest and detected by enhanced chemiluminescence technique (PerkinElmer).
4
Co-immunoprecipitation and Western Blotting of STIM1 and Dynein
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To co‐immunoprecipitate and analyze by Western blotting STIM1 constructs and the dynein–dynactin complex components, ECs or HEK293Ts were lysed in buffer containing 25 mM Tris–HCl pH 7.2, 150 mM NaCl, 1% NP‐40, 5% glycerol, 5 mM MgCl2, 1 mM PMSF, 1 mM Na3VO4, and protease inhibitor cocktail.
Cellular lysates were incubated for 20 min on wet ice and then centrifuged at 15,000 g, 20 min, at 4°C. The total protein amount was determined using the bicinchoninic acid (BCA) assay (Pierce). Equivalent amounts (1 mg) of protein were immunoprecipitated for 2 h at 4°C with the antibody of interest, and immune complexes were recovered on protein A‐ or G‐Sepharose (GE Healthcare) for 1 h at 4°C. Where indicated in the figure legend, cell lysates were pre‐cleared on Sepharose beads before incubation with the antibody of interest. Controls of immunoprecipitations were performed incubating cell lysate with empty immunoglobulin (Ig) G of the same specie of the antibody of interest (Normal rabbit, NI#01, or mouse, NI#03, IgG from Merck Life Science). Immunoprecipitates were washed four times with lysis buffer with or without detergent and then separated by SDS–PAGE. Proteins were then transferred to a Hybond‐C extra nitrocellulose membrane (Amersham), probed with antibodies of interest, and detected by enhanced chemiluminescence technique (PerkinElmer).
Cellular lysates were incubated for 20 min on wet ice and then centrifuged at 15,000 g, 20 min, at 4°C. The total protein amount was determined using the bicinchoninic acid (BCA) assay (Pierce). Equivalent amounts (1 mg) of protein were immunoprecipitated for 2 h at 4°C with the antibody of interest, and immune complexes were recovered on protein A‐ or G‐Sepharose (GE Healthcare) for 1 h at 4°C. Where indicated in the figure legend, cell lysates were pre‐cleared on Sepharose beads before incubation with the antibody of interest. Controls of immunoprecipitations were performed incubating cell lysate with empty immunoglobulin (Ig) G of the same specie of the antibody of interest (Normal rabbit, NI#01, or mouse, NI#03, IgG from Merck Life Science). Immunoprecipitates were washed four times with lysis buffer with or without detergent and then separated by SDS–PAGE. Proteins were then transferred to a Hybond‐C extra nitrocellulose membrane (Amersham), probed with antibodies of interest, and detected by enhanced chemiluminescence technique (PerkinElmer).
5
Endogenous LPHN2 Immunoprecipitation and Western Blot
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To immunoprecipitate and analyze by Western blot endogenous LPHN2, 5 × 106 HUVEC were transferred to ice, washed three times in cold PBS 1X, and surface-labeled at 4°C with 0.2 mg/ml sulfo-NHS-SS-biotin (Thermo Fisher Scientific) in PBS 1X for 30 min. Cells were then washed three times and lysed with a buffer containing 25 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 5% glycerol, 0.5 mM EGTA, 2 mM MgCl2, 1 mM PMSF, 1 mM Na3VO4, and protease inhibitor cocktail (Sigma-Aldrich). Cellular lysates were incubated for 20 min on ice and then centrifuged at 15,000 g, 20 min, at 4°C. The total protein amount was determined using the bicinchoninic acid protein assay reagent (Thermo Fisher Scientific). Equivalent amounts (1 mg) of protein were precipitated for 1 h at 4°C with protein A–Sepharose beads and then centrifuged for 1 min, 15,000 g, at 4°C. Protein A–Sepharose beads were collected to prepare the preclearing samples, and the lysates were then immunoprecipitated for 1 h at 4°C with the rabbit pAb anti-LPHN2 or preimmune serum. Immunoprecipitates were washed four times with lysis buffer and then separated by SDS-PAGE. Proteins were then transferred to a nitrocellulose membrane (Bio-Rad), probed with Abs of interest, and detected by enhanced chemiluminescence technique (PerkinElmer).
6
Protein Extraction and Analysis via SDS-PAGE
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Cells were lysed in buffer containing 25 mM Tris–HCl, pH 7.4, 100 mM NaCl, 5% glycerol, 0.5 mM EGTA, 2 mM MgCl2, 1 mM PMSF, 1 mM Na3VO4, protease inhibitor cocktail (Sigma-Aldrich), and detergent 1% NP-40. Cellular lysates were incubated for 20 min on ice, and then centrifuged at 15,000 g, 20 min, at 4°C. The total protein amount was determined using the bicinchoninic acid protein assay reagent (Thermo Fisher Scientific). Specific amounts of protein were separated by SDS–PAGE with precast Bolt 4–12% Bis-Tris gel (Thermo Fisher Scientific) or Mini PROTEAN TGX precast 7.5% gel (Bio-Rad). Proteins were then transferred to a nitrocellulose membrane (Bio-Rad), probed with Abs of interest, and detected by enhanced chemiluminescence technique (PerkinElmer).
7
HIF-1α Protein Expression Analysis
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Total cell lysate was extracted from HCC cells using RIPA cell lysis buffer (Cell signaling, Danver, MA, USA) according to the manufacturer’s instructions. HIF-1α was bounded with mouse anti-HIF-1α (Cell signaling, Danver, MA, USA) using IgG magnetic beads (Novex, Oslo, Norway), according to the manufacturer’s instructions. The dimerized proteins were detected according to western blot, and blotted with the other target-mouse monoclonal antibodies-HIF-1α (Bethyl, Montgomery, TX, USA) specific for the proteins of interest. The blots were developed using the enhanced chemiluminescence technique (PerkinElmer, Boston, MA, USA) according to the manufacturer’s instructions, and the level of expression of each protein was quantified and compared.
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