PMN-/M-MDSCs were extracted into cold lysis buffer containing 50mM Tris-HCl (pH 7.5), 150 mM NaCl, 1mM EDTA, 1mM MgCl2, 0.5% Triton X-100, phosphatase inhibitor mix (1mM NaF, 1 mMNa3VO4 and 1mM β-glycerol phosphate), and protease inhibitor mix (1mM PMSF, 2μg/mL Roche (Cocktail: Inhibitor Protease aprotinin, 1μg/mL leupeptin, and 1μg/mL pepstatin A). Complete Cell lysates were clarified by centrifugation at 12,000×gcontent for 15min and subjected to SDS-PAGE (8-10% polyacrylamide gels), transferred onto PVDF membranes (Millipore, Bedford, MA). The membrane was blocked in TBS-T buffer (20mM Tris-Hcl, pH 7.5, 150mM NaCl and 0.05% Tween-20) containing 5% (w/v) non-fat milk at room temperature (22°C) for 1 h and then probed with antibodies for total and phosphorylated AMPKα (Cell Signaling Technology), total HIF-1α (Cell Signaling Technology, D2U3T) and GAPDH (Santa Cruz Biotech) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated anti-IgG for 1h at room temperature. Detection was performed with the SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit (Pierce, Rockford, IL, USA). The band images were digitally captured and quantified with a Fluor Chem FC2 imaging system (Alpha Innotech, San Leandro, CA, USA).
Total hif 1α
Manufactured by Cell Signaling Technology
Total HIF-1α is a laboratory reagent used to quantify the total amount of Hypoxia-Inducible Factor-1 alpha (HIF-1α) protein in cellular samples. HIF-1α is a transcription factor that regulates the expression of genes involved in the cellular response to hypoxia. The Total HIF-1α reagent provides a reliable and reproducible method for measuring HIF-1α levels, which is useful for studying the cellular response to various environmental and experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Fluorchem fc2 imaging system, by Bio-Techne (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Mct 4, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Sea block blocking buffer, by Thermo Fisher Scientific (1 mentions)
2 protocols using total hif 1α
1
Western Blot Analysis of AMPK and HIF-1α
2
Metabolic Regulators and Cell Death Analysis
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Cells were seeded at a density of 5.0 × 105 cells/well into 6 wells microplates (BD Biosciences, San Jose, CA) and incubated at 37°C and 5% CO2. After 24 hours, cells were treated with 25 mM MDN-0066 and with DMSO as a negative control and incubated at 37°C for 24 or 48 hours. Cells were lysed in cell lysis buffer (Cell Signaling Technology, Danvers, MA). Twenty to fifty micrograms of protein (as determined by Pierce BCA Protein Assay Kit, Thermo Scientific) were resolved on 12% SDS-polyacrylamide gels and then transferred onto to a pre-wetted in methanol PVDF membrane that was blocked using SEA BLOCK Blocking Buffer (Thermo Scientific). Total HIF-1α, MCT4, C-Myc, Beclin, LC3b, LDHA, cleaved PARP (Cell Signaling Technology, Danvers, MA) and PKM2 (ProteinTech, Chicago, Illinois) were used as primary antibodies. β-actin (Sigma-Aldrich, St Louis, MO) was used as housekeeping protein for normalization. Anti-mouse and anti-rabbit DyLight 700 and 800 conjugated (Cell Signaling Technology, Danvers, MA) were used as secondary antibodies. Membrane was washed and read in Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, Nebraska USA).
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