Western blot analyses with whole-cell RIPA buffer protein lysates were separated by SDS-PAGE and blotted onto Hybond-C-Extra nitrocellulose membranes (Amersham Bioscience, Little Chalfont, UK) or onto Trans-Blot Turbo Mini PVDF membranes (BioRad). Following primary antibodies were used after blocking: anti-HIF-1α (1:500, rabbit polyclonal, Millipore, CA) anti-HIF-2α (1:500, rabbit polyclonal Abcam), anti-c-Myc (1:100, mouse monoclonal Santa Cruz), anti-GLS (1:500, rabbit polyclonal, Proteintech), anti-SDHA (1:2,000, mouse monoclonal Abcam) and anti-β-Actin (1:500, mouse monoclonal Santa Cruz). The proteins were detected by using HRP-conjugated secondary antibodies; anti-mouse IgG (1:5,000, GE healthcare, Buckinghamshire, UK) and anti-rabbit IgG (1:5,000, GE healthcare) and EZ-ECL chemiluminescence detection kit (Biological Industries, Israel).
Anti hif 1α
Manufactured by Merck Group
Sourced in United States, Germany
Anti-HIF-1α is a laboratory reagent that specifically binds and inhibits the HIF-1α (Hypoxia-Inducible Factor-1 alpha) protein. HIF-1α is a transcription factor that plays a central role in the cellular response to low oxygen levels (hypoxia). Anti-HIF-1α can be used in research applications to study the function and regulation of HIF-1α in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Hybond c extra nitrocellulose membrane, by Cytiva (1 mentions)
Trans blot turbo mini pvdf membrane, by Bio-Rad (1 mentions)
Anti gls, by Proteintech (1 mentions)
Anti sdha, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti hif 2α, by Abcam (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Ez ecl chemiluminescence detection kit, by Sartorius (1 mentions)
Hybond, by Cytiva (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Vimentin, by Merck Group (1 mentions)
N cadherin, by Merck Group (1 mentions)
E cadherin, by Merck Group (1 mentions)
Twist, by Merck Group (1 mentions)
Anti cdc25c, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti α sma, by Merck Group (1 mentions)
18 protocols using anti hif 1α
1
Western Blot Protocol for Hypoxia Regulators
2
Protein Expression Analysis by Western Blot
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Cells were lysed on ice in lysis buffer [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate] (Invitrogen, Camarillo, CA, USA) supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Whole cell extracts (20 µg/lane) were electrophoresed using 10% SDS-polyacrylamide gels and electrotransferred to polyvinylidene fluoride membranes (Millipore, Schwalbach, Germany) as described previously.3 (link) The following antibodies were used for Western blotting: anti-HIF-1α (1:1000), -E-cadherin (1:500), -N-cadherin (1:500), -vimentin (1:1000), -Twist (1:1000), -Slug (1:1000), and GAPDH (1:1000). All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
3
Western Blotting and Co-IP Analyses
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For western blotting and Co-IP analyses, cells pellets were lysed by using modified RIPA buffer (5 mM EDTA, 2 mM Na3VO4, 5 mM NaF and 1 mM PMSF) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). For western blotting analysis, SDS-PAGE was performed, and proteins were electroblotted onto PVDF membranes. Co-IP analyses were performed using 300 μg proteins from whole cells lysates. Samples were incubated overnight in IP buffer (250 mM NaCl, 15 mM MgCl2, 40 mM Hepes, 60 mM glycerophosphate) additioned with protease inhibitors with primary antibody in presence of CNRr-activated sepharose 4B (Pharmacia), resolved by SDS-PAGE, transferred into PVDF membranes and labeled with anti-HIF-1α and/or anti-p300 primary antibodies. The primary antibodies used for Immunoblotting were purchased from: Novus Biologicals, (anti-HIF-1α), Millipore™ (anti-p300 and anti-Cyclin A), Santa Cruz (anti-Cdc25C, anti-Cyclin E and anti-β-Actin), Cell Signaling Technology® Inc (anti-Cyclin B1, anti-Cyclin D1, anti-Caspase 3 and anti-Caspase 9) and Oncogene Research (anti-p53). The secondary antibodies used for Immunoblotting were purchased from: GE Healthcare (anti-rabbit and anti-mouse) and Santa Cruz (anti-goat).
4
Western Blot Analysis of Cellular Markers
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Western blot analysis was performed essentially according to an established procedure [18 (link)]. The primary antibodies used were as follows: anti-β-actin (Sigma, St. Louis, MO), anti-α-SMA (Sigma, St. Louis, MO), anti-E-cadherin (BD Transduction Laboratories, Lexington, KY), anti-fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HFE (Santa Cruz Biotechnology, Santa Cruz, CA), anti-vimentin (Boster, Wuhan, China), and anti-HIF-1α (Millipore, USA). Quantifications were performed by measuring band intensities using Image J analysis software.
5
Western Blot Analysis of IPO7, HIF-1α, and Actin
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Total cell lysates or nuclear/cytoplasm fractions were resolved in 6 or 8% SDS-PAGE and analyzed by Western Blot. Primary antibodies used in the experiments were: anti-IPO7 (1:500; ThermoFischer), anti-HIF-1α (1:1000; Millipore) and anti-actin beta (1:500; Cell Signaling Technology, Beverly, MA). Secondary antibodies (anti-mouse or anti-rabbit depending on primary antibody) were Alexa Fluor or HRP conjugated. When HRP conjugated secondary antibodies were used, protein bands were detected using Amersham ECL Western Blotting Detection Reagent; protein bands were finally visualized using Chemidoc (BioRad). The signal intensity of each band was calculated with Image J software (http://rsbweb.nih.gov/ij/ ).
6
Evaluating HIF-1α and C/EBP-β Interaction
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Cells were lysed in the MLB buffer (125 mM Tris-HCl, 750 mM NaCl, 1%v/v NP40, 10%v/v glycerol, 50 mM MgCl2, 5 mM EDTA, pH 7.5, supplemented with the protease inhibitor cocktail set III (Sigma Aldrich), 25 mM NaF, 1 mM NaVO4, 10 mg/ml aprotinin), sonicated and centrifuged at 13000×g for 10 minutes at 4 °C. 50 μg of proteins were subjected to immunoblotting and probed with the following antibodies: anti-HIF-1α (clone 54, BD Transduction Laboratories), C/EBP-β (clone C-19, directed against the common C-terminus of LIP and LAP, Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by a peroxidase-conjugated secondary antibody. Anti-actin (clone C-4, Sigma Aldrich) was used as a control of equal protein loading. The proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). In co-immunoprecipitation assays, 100 μg of whole cell lysates were immuno-precipitated at 4 °C overnight with the PureProteome Protein A/G Mix Magnetic Beads (Millipore) as per manufacturer’s instruction, in the presence of the anti-C/EBP-β antibody (clone C-19, diluted 1/50), then blotted with the anti-HIF-1α antibody as reported above.
7
Western Blot Analysis of Inflammatory Markers
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HOK cells and mucosal epithelia were denatured in laemmli buffer, centrifuged and then heated for 10 min at 95 °C. Proteins were separated by SDS-PAGE, and then electroblotted onto PVDF membranes (Millipore). Western blot analyses were carried out as described [32 (link)]. The following primary antibodies (1:1000 dilution) were used in this study: anti-IKKβ, anti-phospho-NF-κB p65, anti-NF-κB p65 from Cell Signaling, anti-Toll-like receptor 4 (TLR4), anti-IFNγ, anti-IL-1β, anti-TNFα, anti-interleukin 6 (IL-6), anti-VDR and anti-β-actin from Santa Cruz Biotechnology, anti-HIF-1α from Millipore, anti-PHD1, anti PHD2, and anti-VHL from Abcam.
8
Phyllanthus Species Protein Analysis
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Protein lysates were prepared for the four Phyllanthus species-treated and -untreated samples. Protein concentrations were determined using a 2D Quant kit from GE Healthcare (Little Chalfont, UK). Briefly, 150 μg of each sample was either resolved by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or by 2D-gel electrophoresis with 7-cm immobilized pH gradient (IPG) gel strips (pH 3–11 NL; GE Healthcare). The proteins were transferred onto a membrane at 250 mA for 1 h, blocked, and incubated with various primary and secondary antibodies. The target proteins were detected using a colorimetric-based 3,3′-diaminobenzidine substrate. Anti-pan-Ras, anti-Bcl-2, anti-HIF-1α, anti-c-Raf, anti-c-Myc, anti-p53, anti-Elk1, anti-c-Jun/activator protein (AP)-1, anti-JNK1/2, anti-VEGF, goat anti-mouse, and goat anti-rabbit IgG peroxidase-treated antibodies were obtained from Merck Millipore (Darmstadt, Germany), while the anti-RSK antibody was obtained from Thermo Fischer Scientific. These proteins play a crucial role in the pathways affected by the four Phyllanthus species as confirmed using the pathway-reporter array.
9
Radioactive Tracer Acquisition Protocol
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Methyl-[3H]-thymidine was purchased from ICN Biomedicals, Inc. (Irvine, CA, USA). 22NaCl, 86RbCl, H36Cl and [14C]-urea were obtained from PerkinElmer (Waltham, MA, USA), Isotope (St. Petersburg, Russia) and Amersham (Montreal, QC, Canada). DEVD-AMC, DEVD-CHO and z-VAD.fmk were procured from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA). Anti-HIF-1α and anti-GAPDH antibodies were sourced from Merck Millipore (Billerica, MA, USA). The remaining chemicals were supplied by Gibco BRL (Gaithersburg, MO, USA), Calbiochem (La Jolla, CA, USA), Sigma and Anachemia (Montreal, QC, Canada).
10
Quantification of VEGF and HIF-1α in HUVECs
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After treatment with HI (0.00-0.93 mg/mL) in both oxygen conditions, aliquots of 1x10 6
HUVECs were fixed with a solution of 4% paraformaldehyde and permeabilized with ice cold 90% methanol. Afterward, cells were incubated for 30 min with anti-VEGF (1:500, Abcam, San Francisco, CA, USA) or anti-HIF-1α (1:500, Merck Millipore) antibodies, washed and incubated with the FITC-conjugated secondary antibody (1:500, Sigma). For VEGFR-2, 1x10 6 cells were stained with 2.5 µL of anti-VEGFR-2-PE Cy-5.5 antibody (2.5:10, Biolegend, San Diego, CA, USA). Mean fluorescence intensity was quantified by flow cytometry through the analysis of 10,000 events/sample. In order to exclude non-specific bindings, the fluorescence of the isotype negative control antibody [FITC Mouse IgG1, k (FC), Biolegend] was analyzed.
HUVECs were fixed with a solution of 4% paraformaldehyde and permeabilized with ice cold 90% methanol. Afterward, cells were incubated for 30 min with anti-VEGF (1:500, Abcam, San Francisco, CA, USA) or anti-HIF-1α (1:500, Merck Millipore) antibodies, washed and incubated with the FITC-conjugated secondary antibody (1:500, Sigma). For VEGFR-2, 1x10 6 cells were stained with 2.5 µL of anti-VEGFR-2-PE Cy-5.5 antibody (2.5:10, Biolegend, San Diego, CA, USA). Mean fluorescence intensity was quantified by flow cytometry through the analysis of 10,000 events/sample. In order to exclude non-specific bindings, the fluorescence of the isotype negative control antibody [FITC Mouse IgG1, k (FC), Biolegend] was analyzed.
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