The protein levels of genes were detected using western blot analysis. After the transfection for 48 h, the protein was extracted using RIPA buffer and separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrolysis gel. Then, the protein was transferred into a PVDF membrane. After 1 h of the block with 5% non-fat milk, the membrane was incubated with primary antibodies at 4°C overnight and subsequently secondary antibodies at room temperature for 1 h. The primary antibodies anti-HIF1α (20960-1-AP) and anti-TRIM14 (15742-1-AP) were purchased from ProteinTech Group (Chicago, IL, United States). Protein bands were developed using an ECL kit (Solarbio Science and Technology Company, Beijing, China).
Anti hif1α 20960 1 ap
Manufactured by Proteintech
Sourced in United States
Anti-HIF1α (20960-1-AP) is a primary antibody product from Proteintech. It is designed to detect the hypoxia-inducible factor 1-alpha (HIF1α) protein.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti mouse igg, by Merck Group (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Ecl kit, by Solarbio (1 mentions)
Anti α tubulin t6074, by Merck Group (1 mentions)
Anti hsl 4107, by Cell Signaling Technology (1 mentions)
Anti lamin b1 ab16048, by Abcam (1 mentions)
Acipimox a7856, by Merck Group (1 mentions)
Anti β actin a3854, by Merck Group (1 mentions)
Ab28479, by Abcam (1 mentions)
Anti cd36 18836 1 ap, by Proteintech (1 mentions)
Anti rabbit igg, by Merck Group (1 mentions)
Anti c ebpα, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Anti myc 60003 2 ig, by Proteintech (1 mentions)
Hrp conjugated anti rabbit igg, by Merck Group (1 mentions)
Anti rhoa arh04, by Cytoskeleton (1 mentions)
Anti ha 3724, by Cell Signaling Technology (1 mentions)
4 protocols using anti hif1α 20960 1 ap
1
Western Blot Analysis of HIF1α and TRIM14
2
Antibody Characterization for Cell Signaling Analysis
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l -Homocysteine (Hcy, H4628), acriflavine (ACF, HY-100575) and acipimox (A7856) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used in this work: anti-ERO1α (MABT376) antibody was purchased from Merck Millipore (Darmstadt, Germany). Anti-HIF1α (20960-1-AP, RRID:AB_10732601 ) and anti-Rab18 (60057–1-Ig, RRID:AB_2173930 ) antibodies were purchased from Proteintech (Rosemont, IL, USA). Anti-ATGL (2138, RRID:AB_2167955 ), anti-HSL (4107, RRID:AB_2296900 ), anti-phospho HSL (Ser660) (4126, RRID:AB_490997 ), anti-phospho HSL (Ser563) (4139, RRID:AB_2135495 ) and anti-GAPDH antibodies (2118L, RRID:AB_561053 ) were purchased from Cell Signaling (Danvers, MA, USA). Anti-Lamin B1 (ab16048, RRID:AB_443298 ) was purchased from Abcam (Cambridge, MA, USA). Anti-α-tubulin (T6074, RRID:AB_477582 ), anti-β-actin (A3854, RRID:AB_262011 ) and peroxidase-conjugated goat anti-mouse IgG (A4416, RRID:AB_258167 ) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and peroxidase-conjugated goat anti-rabbit IgG (ZB2301, RRID:AB_2747412 ) was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China).
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3
Quantifying Cell Signaling Proteins via Western Blot
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Antibodies were obtained from the following companies: anti-CNF1 (sc-52655, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LXRβ (ab28479, Abcam), anti-C/EBPα (8178, Cell Signaling Technology, Danvers, MA, USA), anti-HIF1α (20960-1-AP, Proteintech, Chicago, IL, USA), and anti-CD36 (18836-1-AP, Proteintech). Whole cell lysates were prepared using RIPA lysis buffer (Millipore, Billerica, MA, USA), adding complete protease inhibitors (Roche, Basel, Switzerland). The protein concentration was determined using the BCA Protein Assay Kit (ThermoFisher, Waltham, MA, USA). Approximately 30 μg of cell lysates were used for loading. Antibody binding was revealed using an HRPA conjugated anti-rabbit IgG (Sigma-Aldrich) or anti-mouse IgG (Sigma-Aldrich). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and exposure to Tanon-5200 machine.
4
Antibody Characterization and Protein Quantification
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Antibodies were ordered from the following companies: anti-HIF1α (20960-1-AP, 1:1000), anti-HSF1 (51034-1-AP, 1:1000), anti-RhoC (10632-1-AP, 1:1000), and anti-Myc (60003-2-Ig, 1:2000) were from Proteintech (Chicago, IL,USA); anti-HSF1 phosphorylated at Ser326 antibody (ab76076, 1:2000) was from Abcam (Cambridge, MA, USA); anti-HA (#3724, 1:1000), and anti-HSP90α (#8165, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA); anti-RhoA (ARH04, 1:500) and anti-Rac1 (ARC03, 1:500) were from Cytoskeleton (Denver, CO, USA); anti-RhoB (sc-8048, 1:500) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Cdc42 (BA2442, 1:500) was from Wuhan Boster Biological Technology Co. Ltd. (Wuhan, China); and anti-Flag (F1804, 1:1000) was from Sigma-Aldrich. Cells were washed with PBS three times after treatment. Whole cell lysates were prepared using RIPA lysis buffer (Merck Millipore), with the addition of complete protease inhibitors (Roche). The protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific) and approximately 20 μg of cell lysates were used. Antibody binding was revealed using an HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma-Aldrich). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and exposed in a Tanon-5200 machine.
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