Human prostate carcinoma LNCaP and 22Rv1 cells were obtained from the American Type Culture Collection (Manassas, VA). LNCaP and 22Rv1 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate at 37°C in a humidified 5% CO2 incubator. Media and other cell culture materials were from Invitrogen Corporation (Gaithersburg, MD). Cells were treated with different concentrations of silibinin (5–200 μM in medium), dissolved originally in dimethyl sulfoxide (DMSO), for descried time periods. An equal amount of DMSO (vehicle) was present in each treatment, including control; DMSO concentration did not exceed 0.1% (v/v) in any treatment. Antibodies for HIF-1α, HIF-1β, PHD1, PHD2, FIH (factor inhibiting HIF), phosphorylated and total ACC, and FASN were from Cell Signaling (Beverly, MA). Ki-67 and CD31 antibodies were from Abcam (Cambridge, MA), cyclin D1 antibody was from Thermo Scientific (Fremont, CA), TUNEL kit was from Promega (Madison, WI), PSA (prostate specific antigen) antibody was from DAKO (Carpinteria, CA), and HIF-1α antibody used for immunohistochemistry (IHC) was from Santa Cruz Biotechnology (Dallas, TX). Enhanced chemiluminescence (ECL) detection system was from GE healthcare (Buckinghamshire, UK). All other reagents were obtained in their commercially available highest purity grade.
Hif 1α antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The HIF-1α antibody is a laboratory tool used to detect and measure the expression of the hypoxia-inducible factor-1 alpha (HIF-1α) protein. HIF-1α is a transcription factor that plays a crucial role in the cellular response to low oxygen levels (hypoxia). The antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to identify and quantify the presence of HIF-1α in biological samples.
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Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Tunel kit, by Promega (1 mentions)
Enhanced chemiluminescence ecl detection system, by GE Healthcare (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
D mannitol, by Merck Group (1 mentions)
Np 40, by Merck Group (1 mentions)
Jnj 10198409, by Merck Group (1 mentions)
Tgf β antibody, by Abcam (1 mentions)
Fugene transfection reagent, by Promega (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Na3vo4, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Anti ha antibody, by Fortrea (1 mentions)
Tgf β, by R&D Systems (1 mentions)
35s methionine, by PerkinElmer (1 mentions)
Mounting media, by Agilent Technologies (1 mentions)
14 protocols using hif 1α antibody
1
Silibinin Inhibits Prostate Cancer Cells
2
Investigating PDGFR-Mediated Signaling Pathways
3
Chromatin Immunoprecipitation Assay for HIF-1α
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The chromatin immunoprecipitation assay was performed as follows. The cell lysate of Hus/L-FABP or control cells was sonicated, and then the chromatin was immunoprecipitated with HIF-1α antibody or rabbit immunoglobulin G antibody (Santa Cruz Biotechnology) as a negative control. After precipitation, the bound DNA was dissolved with 40 μl of ddH2O and then amplified by PCR with primers amplifying the HIF-1α binding element (−1041 to −750, Supplementary Table 1 ). The final PCR products were analyzed using 1.8 % agarose gels and visualized by ethidium bromide staining.
4
HIF-1α Immunoprecipitation Assay
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HeLa cells were lysed in radioimmunoprecipitation buffer as aforementioned. About 300 μg of cell lysates were used per immunoprecipitation condition. Protein lysates were incubated with 2 μg of HIF-1α antibody (Santa Cruz; sc-53546) or 2 μg of mouse IgG control antibody (Sigma) in a rotating platform at 4 °C overnight. About 20 μl of packed protein-G-sepharose beads (Pierce) were used to recover the immunocomplexes, by incubation in a rotating platform for 1.5 h at 4 °C. Beads were washed with 1× PBS buffer thrice. The complexes were eluted from beads with SDS-loading buffer and resolved as described previously by immunoblotting.
5
Evaluating Hypoxia-Induced Signaling in A549 Cells
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A549 cells were seeded in 8‐chambered glass plates. After 24 h incubation, cells were treated with HCl (pH 4.0, DMEM) for 30 min at 37°C in 5% CO2. Control cells were exposed to phosphate‐buffered saline (PBS). After incubation with HCl, the acidified medium was discarded, and the cells were washed thrice with complete media to confirm neutralization. Cells were fixed by adding formaldehyde directly to the cells for 15 min at room temperature. Cells were washed with phosphate‐buffered saline (PBS), permeabilized with 0.2% Triton X‐100 (Sigma‐Aldrich), washed again with PBS twice, and blocked with PBS containing 1% bovine serum albumin (BSA). This cell was followed by incubation in HIF‐1α antibody (Santa Cruz Biotechnology) and NF‐kBp65 (Cell Signaling) followed by incubation in Alexa flour labeled secondary antibodies (Invitrogen) for 1 h. Nuclei were stained with DAPI. The cells on coverslips were then mounted in mounting media (Dako), and photomicrographs of the invasive sections were analyzed.27 , 51
6
Biotinylation and Immunoblotting of ER and HIF-1α
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PC3 cells were transfected with 3 μg BirA (biotin ligase) expression plasmid, 3 μg plasmid containing biotinylation consensus tagged receptors B7TEV-ERα, B7TEV-ERβ1, B7TEV-ERβ2 and B7TEV-ERβ5 together with 3 μg pcDNA3 HA-HIF-1α “Addgene plasmid 18949” or pcDNA3 HA-HIF-1α P405/A-P564/A “Addgene plasmid 18955” or pcDNA3 HA-HIF-1α Δ401–603 (pcDNA3 HA-HIF-1αΔODD) described by Kondo et al and Yan et al. [27 (link), 28 (link)] in a 100 mm tissue culture plate. For HEK293 cells the biotin ligase was transiently transfected. After 48 h, cells were scraped, pelleted and lysed in 300 μl NETN (20 mM Tris (pH = 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), briefly sonicated and centrifuged to remove debris. 10μl streptavidin magnetic beads (Pierce Rockford, IL) were washed in NETN and incubated with 300 μl cellular extract for 2 hours in the cold room. Beads were washed (3 x 10 minutes rotation) with 300 μl NETN and boiled with 20 μl of 2× sample buffer [125 mM Tris HCl (pH 6.8), with 4% SDS, 20% (vol/vol) glycerol, and 0.004% bromophenol blue] and subjected to SDS-PAGE and transferred to nitrocellulose membrane for Western blot. The blot was probed with HIF-1α antibody (Santa Cruz Dallas, TX) 1:1000 dilution and secondary antibody 1:10,000 dilution.
7
ChIP-PCR Analysis of HIF-1α Binding
8
Western Blot Analysis of HIF-1α and VEGF
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Total cell lysates were prepared by harvesting cells in protein extraction buffer, and protein concentration was analyzed using the BCA protein assay kit. Equal amounts of proteins were separated on 6% or 10% SDS-polyacrylamide gel, transferred onto nitrocellulose membranes (Schleicher and Schuell Bio-Science). The membranes were blocked with 5% nonfat milk in TBST (Tris-buffered saline, pH 7.4 and 0.05% Tween 20) and incubated with HIF-1α antibody (1:200), VEGF antibody (1:250) and β-actin antibody (1:250) (all from Santa Cruz Biotechnology Inc, CA, USA) overnight at 4°C. The membranes were then incubated with alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:2000) for 1 h at room temperature, and the signals were detected by using enhanced chemiluminescence detection kit.
9
ChIP Assay of m6A and HIF1α Targets
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ChIP assay involved using the Simple Chip Enzymatic Chromatin immunoprecipitation kit (Cell Signaling Technology, USA) based on the manufacturer’s protocol. Briefly, treated HUVECs were first cross-linked with 1% formaldehyde, quenched by glycine, then digested with micrococcal nuclease. An amount of 2% lysates was used as an input reference. The lysates were incubated with 5 μg anti-m6A antibody (EMD Millipore ABE572, USA), HIF1α antibody (Santa Cruz Biotechnology, CA, USA) or goat anti-mouse IgG (ab205719, Abcam, USA) for immunoprecipitation. Then immunoprecipitates were treated with Protein G Agarose Beads overnight at 4 °C with gentle shaking. The immunoprecipitated DNA samples were cross-linked reversed, purified and amplified by PCR with their specific primers (Table S1 ).
10
Astragalus-Based Herbal Formula for Cancer
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WPX comprises the following components: Huangqi (Astragalus Membranaceus)30 g, Taizishen (Pseudostellaria Heterophylla)15 g, Baishu (Atractylodis Macrocephalae)15 g, Eshu (Curcuma zedoaria)10 g, Danshen (Salvia Miltiorrhiza)10 g and Baihuasheshecao (Hedyotis Diffusa Willd)30 g. The herbs were provided and authenticated by the First Affiliated Hosipital of Guangzhou University of Chinese Medicine. The medical herbs were boiled with distilled water, and concentrated into a mixture containing crude drugs 1.5 g/mL. MNNG was supplied by Tokyo Kabushiki Kaisha, Japan (No. ZG4T1-FP). CD34 antibody was abtained from R&D Systems, USA (lot ZDP0112111); VEGF antibody was supplied by Abcam, UK (lot GR-116031-1); HIF-1α antibody was purchased from Santa Cruz Biotechnology, USA (lot L1212). Maxima™ SYBR Green/Fluorescein qPCR Master Mix (2X) was supplied by Fermentas, USA.
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