Cells and tissue samples were lysed in RIPA buffer with PMSF as protease inhibitor (Beyotime, Shanghai, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on NC or PVDF membrane (Millipore, Bedford, MA). After blocking with 5% dry milk in TBST for 2 h at room temperature, the membranes were incubated with the primary antibodies against SDHB (1:1000, Epitomics), β-tubulin (1:4000, Epitomics), β-actin (1:500, Abmart), caspase 3 (1:1000, Cell Signalling Technology), Bcl-2 (1:4000, Epitomics), MMP-2 (1:500, Abcam), FAK (1:1000, CST), p-FAK (1:1000, CST), AMPKα (1:1000,CST), p-AMPKα (1:1000, CST), GAPDH (1:4000, Abmart), P38 (1:1000, CST), p-P38 (1:1000, CST), ERK (1:1000, CST), p-ERK (1:1000, CST), HIF-1α (1:1000, Epitomics) in dilution buffer overnight at 4°C. Membranes were washed for three times with TBST, then were incubated with IRDye 800CW conjugated goat (polyclonal) anti-Rabbit IgG or anti-Mouse IgG (1:10000) antibodies for 1 h at room temperature. The expression of specific proteins was detected through the use of Odyssey system following the manufacturer's instructions.
Hif 1α
Manufactured by Abcam
Sourced in United States, United Kingdom, China, France, Germany
HIF-1α is a transcription factor that plays a key role in the cellular response to hypoxia. It is the oxygen-regulated subunit of the HIF-1 complex, which activates the transcription of genes involved in adapting to low oxygen conditions.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (29 mentions)
Pvdf membrane, by Merck Group (24 mentions)
Vascular endothelial growth factor (vegf), by Abcam (20 mentions)
β actin, by Merck Group (19 mentions)
Gapdh, by Abcam (19 mentions)
Hif 2α, by Abcam (15 mentions)
β actin, by Santa Cruz Biotechnology (12 mentions)
Vegfa, by Abcam (12 mentions)
Glut1, by Abcam (12 mentions)
Bca protein assay kit, by Beyotime (12 mentions)
Ripa buffer, by Thermo Fisher Scientific (12 mentions)
Gapdh, by Proteintech (11 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
P akt, by Abcam (9 mentions)
P erk, by Cell Signaling Technology (9 mentions)
Vimentin, by Abcam (9 mentions)
E cadherin, by Abcam (9 mentions)
N cadherin, by Abcam (8 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
Hif 2α, by Novus Biologicals (7 mentions)
294 protocols using hif 1α
1
Western Blot Analysis of Cellular Proteins
2
Quantifying Hypoxia-Induced Metabolic Shifts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed on ice immediately after removal from an incubator with TNG cell lysis buffer (Cell Signaling) containing complete Mini protease inhibitor (Roche). Proteins were separated by electrophoresis on Novex 4–12% Bis-Tris gels (Invitrogen), and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked in 10% milk in PBS with Tween 20, except when being probed for HIF-1α, in which case 5% milk TBS solution with Tween 20 was used. The following antibodies were used to probe the membranes: hypoxia inducible factor-1α (HIF-1α) (Epitomics, 1∶500), lactate dehydrogenase subunit M (LHD-M) (Santa Cruz Biotechnology, 1∶1000), pyruvate dehydrogenase kinase 1 (PDHK1) (Cell Signaling, 1∶1000), ATP synthase subunit β (ATP5β) (Abcam, 1∶1000), carbonic anhydrase nine (CAIX) (Abcam, 1∶1000), and β-actin (Sigma Aldrich, 1∶30,000). Densitometry was performed using ImageJ.
3
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed twice with PBS and resuspended in lysis buffer (50 mM Tris–HCl pH 8.0, 1 mM EDTA, 250 mM NaCl, 1% NP-40 and 0.5% Na-Deoxycholate). Cell lysates were then shaken for 30 min on an orbital shaker at 4 °C and centrifuged for 20 min at 12,000×g and the protein containing supernatant was collected. Protein concentration of cell lysates was estimated using a commercial kit (Bio-Rad, USA). SDS-PAGE and Western blot were performed to determine the expression of Arg-1 (GTX109242, Gene Tex, USA), iNOS (GTX31048, Gene Tex, USA), Hif-1α (2015–1, Epitomics, USA), IRAK1 (4504, Cell signaling, USA), IRAK2 (4367, Cell signaling, USA), IRAK4 (4363, Cell signaling, USA), IRAK-M (4369, Cell signaling, USA), pERK1/2 (2219–1, Epitomics, USA), pSTAT1 (7649, Cell signaling, USA), pSTAT6 (9361, Cell signaling, USA), STAT1 (10144–2-AP, Proteintech, USA), STAT6 (51073–1-AP, Proteintech, USA), VEGF (sc-7269, Santa cruz, USA), β-actin (M2010S, Abmart, China).
4
Immunohistochemical Analysis of PDAC Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To expand the numbers of patients in the sample set, tissue microarrays were constructed from the archival formalin-fixed paraffin-embedded tissue blocks of 120 surgically resected primary PDACs. Immunohistochemistry was performed as previously described (Muders et al, 2006 (link)). Primary antibodies were OS-9 (1 : 100; Novus Biologicals) and HIF-1α (1 : 250; Epitomics, Burlingame, CA, USA). Scoring of the immunohistochemical staining was based on the percentage of positive cells and staining intensity under a light microscope at × 200 magnification. Four categories were denoted (0, 1, 2 and 3) as negative, weak, moderate and strong. Groups 2 and 3 were considered high-expression groups and groups 0 and 1 as low-expression groups.
5
Chromatin Immunoprecipitation Assay for Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin was extracted from cells (5 × 106) and ChIP assays were performed as previously described [18 (link)]. The differential binding between proteins and promoter DNA was examined by PCR. The primary Abs used were as follows: HIF-1α (Abcam, UK), β-arr1 (Santa Cruz Biotechnology CA, USA), p300 (Santa Cruz Biotechnology CA, USA) and acetyl-Histone 3 (AcH3) (BD Laboratory Transduction, NJ, USA). The primers used were as follows: ET-1 promoter Fw: 5′-CAGCTTGCAAAGGGGAAGCG-3′ and Rev: 5′-TCCGACTTTATTCCAGCCCC-3′; VEGF promoter Fw: 5′-AGGAACAAGGGCCTCTGTCT-3′ and Rev: 5′-CAGTGTGTCCCTCTGACAATG-3′.
6
Protein Expression Profiling of C2C12 Myotubes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C2C12 myotubes were lysed in RIPA buffer containing protease inhibitor and PMSF to extract the total protein. Equal quantities of proteins (20 μg) were separated by 10%–12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies targeting HIF-1α (1 : 1000; Abcam, Cambridge, UK), BNIP3 (1 : 1500; Abcam), atrogin-1 (1 : 1000; Abcam), LC3B (1 : 1000; ABclonal, Woburn, MA), beclin-1 (1 : 2000; ABclonal), myogenin (1 : 500; Millipore, Billerica, MA), parkin (1 : 1000; CST, Danvers, MA), p62 (1 : 500; CST), p-mTOR (1 : 1000; CST), mTOR (1 : 1000; CST), p-AMPKα (1 : 1000; CST), and AMPKα (1 : 1000; CST) overnight at 4°C. The membranes were incubated with goat anti-mouse or anti-rabbit secondary antibody for 1 hour at room temperature. Band intensity was determined using a chemiluminescent imaging system (Tanon, Shanghai, China). Tubulin was used as a control for protein level quantification.
7
Immunofluorescence Assay for HIF-1α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde, washed with PBS, and then blocked with10% goat serum. They were then incubated with HIF-1α (1:200, Abcam) antibodies overnight at 4 °C. After incubation, cells were washed twice with PBS and stained with Cy3 (red)-conjugated secondary antibody for a further 2 h at 37 °C. Surplus antibody was removed by washing before obtaining images with an Olympus microscope (Olympus, Tokyo, Japan) at × 40 magnification. Images were captured with a DP50 camera and DP50 software (Olympus).
8
Western Blot Analysis of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To extract proteins for western blot analysis, cells were first lysed in RIPA buffer supplemented with proteinase inhibitors (Pierce Biotechnology, Rockford, IL, USA). Equivalent concentrations of protein measured by a BCA Protein Assay Kit (Thermo Fisher Scientific) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% non-fat milk for 1 h and then incubated overnight at 4 °C with primary antibodies against Tie1 (1:1000, Abcam, Cambridge, UK), BMI-1 (1:1000, Abcam), LGR5 (1:1000; Bioss, Beijing, China), CD44 (1:1000, Cell Signaling Technology, Danvers, MA, USA), HIF-1α (1:2000, Abcam), β-actin (1:5000, Abcam), and HA-tag (1:3000, Proteintech, Wuhan, China). Following incubation for 1 h with HRP-conjugated secondary antibodies (Cell Signaling Technology), immunoreactive bands were visualized with an enhanced chemiluminescence detection system (Thermo Fisher Scientific). The relative intensities of target proteins were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
9
Hypoxia-induced HIF-1α Regulation in MPNST
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytoplasmic and nuclear extractions of MPNST cell lines were prepared separately using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA). To inhibit the degradation of HIF-1α overexpressed under hypoxic conditions for 24 h, the cells were scraped and collected immediately after removal from the hypoxic chamber. Cellular extractions stimulated with chetomin or deferoxamine (DFO) for 24 h were also collected. Western blot analysis was performed as described previously [32 (link), 33 (link)] with the following primary antibodies: HIF-1α (1:1000, Abcam), β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), and lamin A/C (1:200, Santa Cruz). Immunoblotting of HIF-1α was performed with nuclear extraction as described previously [34 (link), 35 (link)].
10
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen heart tissue was pulverized in liquid nitrogen and resuspended in lysis buffer containing fresh protease and phosphatase inhibitors. Equal amounts of protein were fractionated by SDS polyacrylamide gel electrophoresis together with molecular weight standards and transferred to nitrocellulose membranes (Protran; Whatman). Membranes were blocked in 5% BSA or nonfat dry milk (in TBS buffer containing 0.1% Tween‐20), followed by incubation with antibodies against p53 (Cell Signaling Technology), Hif1α (Abcam), or Vegf (Millipore). Protein bands were visualized using horseradish peroxidase‐conjugated secondary antibodies (Amersham Biosciences), followed by detection with SuperSignal West Pico Substrate (Pierce). Protein bands were quantified by densitometry and normalized to Gapdh (HyTest Ltd) or β‐actin (Millipore) protein, and are expressed as ‐fold change versus sham‐operated mice (set at 1).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!