rabbit anti hif1α primary antibody  (Proteintech)

Journal: Redox Biology

Article Title: Functional hypoxia reduces mitochondrial calcium uptake ☆

doi: 10.1016/j.redox.2024.103037

Figure Lengend Snippet: Mitochondrial Ca 2+ uptake was lower under functional hypoxia in mouse myotubes. A) Culture conditions. B) Partial pressure of O 2 in culture media upon exposure to 1 % O 2 in the gas phase ( N = 3). C) HIF1α protein content in myotubes 120 min after exposure to 1 % O 2 in the gas phase ( N = 4). Representative blot of HIF1α with ponceau (loading) stain in myotubes 120 min after exposure to 1 % O 2 in the gas phase ( N = 4). E) Hypoxia and glycolysis hallmark gene sets enriched in myotubes 120 min after exposure to 1 % O 2 compared with 5 % O 2 in the gas phase ( N = 6). False discovery rate FDR. Normalized enrichment score NES. F) TMRM mean intensity under 5 % ( t = 0 min) and after 120 min of exposure to 1 % O 2 in the gas phase ( t = 120 min; N = 4) in C2C12 myotubes. G) Representative images of C2C12 myotubes stained with TMRM exposed to 5 % or 120 min after exposure to 1 % O 2 in the gas phase. Scale bar is 50 μm. An increase in the TMRM intensity indicates a decrease in the mitochondrial membrane potential difference. H) Quantification of Fluo-4AM mean intensity in response to caffeine stimulation in myotubes 120 min after exposure to 5 % or 1 % O 2 in the gas phase ( N = 5). I) Representative images of C2C12 myotubes stained with Fluo-4AM, exposed to 5 % or 1 % O 2 and stimulated with caffeine. Scale bar is 50 μm. J) Response of Rhod-2AM fluorescence to caffeine stimulation in C2C12 myotubes 120 min after exposure to 5 % or 1 % O 2 in the gas phase ( N = 4). K) Representative images of C2C12 myotubes stained with Mitotracker Green and Rhod-2AM, exposed to 5 % or 1 % O 2 and stimulated with caffeine. Scale bar is 20 μm. In panels B, C, F, H and J, the bars represent the means, error bars the standard deviations, dots are individual values.

Article Snippet: The lysates were then processed as described above using an anti-rabbit HIF1α primary antibody (Cayman Chemicals).

Techniques: Functional Assay, Staining, Membrane, Fluorescence

Journal: Redox Biology

Article Title: Functional hypoxia reduces mitochondrial calcium uptake ☆

doi: 10.1016/j.redox.2024.103037

Figure Lengend Snippet:

Article Snippet: The lysates were then processed as described above using an anti-rabbit HIF1α primary antibody (Cayman Chemicals).

Techniques: Recombinant, Software, Control, Isolation, Real-time Polymerase Chain Reaction, Glo Assay, Staining, Blocking Assay, Imaging, Bicinchoninic Acid Protein Assay, DC Protein Assay

Journal: Frontiers in Physiology

Article Title: A novel OSA-related model of intermittent hypoxia in endothelial cells under flow reveals pronounced inflammatory pathway activation

doi: 10.3389/fphys.2023.1108966

Figure Lengend Snippet: Intermittent low grade hypoxia (pcO 2 35 mmHg 15 cycles per hour, for 4 h) does not trigger HIF1α and HIF2α stabilization, but activates sensitive hypoxic molecular probe. After 48 h of 15 dyn/cm 2 shear stress endothelial cells were treated with 5 or 15 cycles per hour of intermittent hypoxia for 4 h. Western Blots of HIF1α and HIF2α (A) did not show any expression in endothelial cell lysates after intermittent hypoxia. In contrast, 5%, and in particular 1% of constant hypoxia or the treatment with Dimethyloxalylglycine (DMOG, 1 mM), an inhibitor for HIF prolylhydroxylases, for 4 h did stabilize HIF1α and HIF2α. Inflammatory activation (TNF 25 ng/mL) resulted in slight HIF expression. However, using a sensitive marker of low dose hypoxia (Image-iT™ green hypoxia reagent), we detected a significant increase in staining following 15 hypoxic cycles per hour in confocal microscopy (B) . Upon completion of confocal images, HUVECs were detached and subjected to detailed fluorescence analysis on a single cell level by flow cytometry. (C) shows a representative histogram of one experiment depicting the mean fluorescence intensity (MFI) of Image-iT™ green hypoxia reagent in all experimental groups. The data of three independent experiments are summarized in the bar graph of (D) . Shown are means ± SEM. n = 3–6, * p < 0.05.

Article Snippet: Non-specific binding was blocked by incubating the membranes with 5% non-fat milk for 1 h. Primary antibodies for HIF1α (#36169), HIF2α (#71565), p-IkB (#2859; all Cell Signaling Technologies), and p-ERK (sc-7383 Santa Cruz Biotec) were diluted in TBST with 5% non-fat milk. β-Actin (#4970; Cell Signaling Technologies) served as the loading control.

Techniques: Shear, Western Blot, Expressing, Activation Assay, Marker, Staining, Confocal Microscopy, Fluorescence, Flow Cytometry

Journal: Molecular Medicine Reports

Article Title: Suppression of CLEC3A inhibits osteosarcoma cell proliferation and promotes their chemosensitivity through the AKT1/mTOR/HIF1α signaling pathway

doi: 10.3892/mmr.2020.10986

Figure Lengend Snippet: Knockdown of CLEC3A suppresses the AKT1/mTOR/HIF1α pathway. (A) Scatter diagram demonstrating the co-expressing relationship between CLEC3A and AKT1 in mRNA level in patient OS tissues. (B) Expression levels of AKT1, mTOR, and HIF1α in the si-NC- and si-CLEC3A-transfected MG63 and SaOS-2 cells was detected using western blotting. (C) Immunofluorescence was used to analyze the nuclear location of HIF1α in the si-NC- and si-CLEC3A-transfected MG63 cells (magnification, ×200). VEGF, GLUT1 and MCL1 mRNA expression levels were detected by reverse transcription-quantitative PCR in the si-NC- and si-CLEC3A-transfected (D) MG62 and (E) SaOS-2 cells. (F) VEGF, GLUT1, and MCL1 protein expression levels were detected in the si-NC- and si-CLEC3A-transfected cells by western blotting.*P<0.05. OS, osteosarcoma; CLEC3A, C-type lectin domain family 3 member A; NC, negative control; si, small interfering RNA; HIF1, hypoxia-inducible factor-1; VEGF, vascular endothelial growth factor; GLUT1, glucose transporter 1; MCL1, induced myeloid leukemia cell differentiation protein Mcl-1.

Article Snippet: The cells were subsequently incubated with rabbit anti-HIF1α primary antibody (1:100; cat no. 20960-1-AP; ProteinTech Group, Inc.) overnight at 4°C.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Immunofluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Small Interfering RNA, Cell Differentiation

Journal: Molecular Medicine Reports

Article Title: Suppression of CLEC3A inhibits osteosarcoma cell proliferation and promotes their chemosensitivity through the AKT1/mTOR/HIF1α signaling pathway

doi: 10.3892/mmr.2020.10986

Figure Lengend Snippet: Restored AKT1/mTOR/HIF1α pathway expression reverses the effect of CLEC3A knockdown on biological functions. (A) Reverse transcription-quantitative PCR was used to detect the mRNA expression levels of VEGF, GLUT1 and MCL1 in si-NC- and si-CLEC3A-transfected MG63 and SaOS-2 cells with or without treatment with SC79. (B) Cell Counting Kit-8 assays were used to detect the rate of cell proliferation in si-NC- and si-CLEC3A-transfected MG63 and SaOS-2 cells with or without treatment with SC79. (C) Colony formation assays were used to detect the colony forming ability in si-NC- and si-CLEC3A-transfected cells with or without treatment with SC79 (diameter of dishes=6 cm). *P<0.05, **P<0.01. CLEC3A, C-type lectin domain family 3 member A; NC, negative control; si, small interfering RNA; VEGF, vascular endothelial growth factor; GLUT1, glucose transporter 1; MCL1, myeloid cell leukemia 1.

Article Snippet: The cells were subsequently incubated with rabbit anti-HIF1α primary antibody (1:100; cat no. 20960-1-AP; ProteinTech Group, Inc.) overnight at 4°C.

Techniques: Expressing, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Cell Counting, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Inhibitory effects of dieckol on hypoxia-induced epithelial-mesenchymal transition of HT29 human colorectal cancer cells

doi: 10.3892/mmr.2016.5872

Figure Lengend Snippet: DK suppressed the HIF1α signaling pathway required for CoCl 2 -induced EMT in HT29 cells. HT29 cells were incubated in the presence or absence of 50 µM CoCl 2 and/or 25 mg/ml DK. Protein expression levels of HIF1α, E-cadherin, vimentin and snail1 in the different treatment groups were analyzed using western blotting. A representative image from three independent experiments has been presented. DK, dieckol; HIF1α, hypoxia-induced factor 1α; EMT, epithelial-mesenchymal transition

Article Snippet: Primary antibodies against HIF1α (cat. no. 610958), E-cadherin (cat. no. 7870), vimentin (cat. no. 5741), Snail1 (cat. no. AV33314) and β-actin (cat. no. MAB1501) were obtained from BD Biosciences, Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), Cell Signaling Technology, Inc. (Danvers, MA, USA), and Sigma-Aldrich; Merck Millipore (Darmstadt, Germany), respectively.

Techniques: Incubation, Expressing, Western Blot