ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free

Immunocytochemistry/ Immunofluorescence: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124]

Immunocytochemistry/Immunofluorescence: ARNT/HIF-1 beta Antibody (H1beta234) [NB100-124] - Co-localization of HIF-2alpha with ARNT/HIF-1 beta antibody (H1beta234) [NB100-124]. ICC/IF detecting indicated proteins using antibodies labelled with Alexa Fluor 555 (red, pseudocolour) and Alexa Fluor 488 (green, pseudocolour). 'Merge' is the red image superimposed onto the green image of the co-stained nuclear proteins. 'Coloc' is the co-localization channel calculated using ImageJ plugin. White indicates pixels where both red and green signal is found (i.e. co-localization). 'Overlay' is the co-localization image superimposed onto the merged image. Inlay is the magnified region (white square). White arrows highlight regions of co-localization. Scale bar, 5 um. Abbreviations: RNAPII, RNA Polymerase II phospho-serine 5. Image collected and cropped by CiteAb from the following publication (https://rsob.royalsocietypublishing.org/lookup/doi/10.1098/rsob.160195), licensed under a CC-BY license.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234)BSA Free [NB100-124]

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) [NB100-124] - Analysis of HIF-1 beta in HeLa nuclear extract using ARNT/HIF-1 beta antibody (H1beta234) [NB100-124]. Theoretical molecular weight 86.6 kDa. Observed molecular weight ~85 kDa.

Immunocytochemistry/ Immunofluorescence: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124]

Immunocytochemistry/Immunofluorescence: ARNT/HIF-1 beta Antibody (H1beta234) [NB100-124] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton X-100. The cells were incubated with ARNT/HIF-1 beta antibody (H1beta234) [NB100-124] at 5 ug/mL overnight at 4C and detected with an anti-mouse DyLight 488 (Green) at a 1:500 dilution. Actin was detected with Phalloidin 568 (Red) at a 1:200 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - PGE1 induces HIF-1 alpha protein accumulation in vascular-derived cells.Human aortic smooth muscle cells (HASMCs) (A & B), human umbilical vein endothelial cells (HUVECs) (C & D), & HEK293 cells (G) were exposed to 1 or 10 µM PGE1 under 20% O2 or 1% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha, HIF-1 beta, & beta-actin protein expression. Experiments were repeated thrice (A, C & G). Representative immunoblots are shown (A & C). Band intensities were analyzed densitometrically (B & D). Fold induction relative to lane 1 was plotted as mean ± S.D. ∗P < 0.05 compared with the control. HASMCs (E) & HUVECs (F) were exposed to 1 µM PGE1 for the indicated times under 20% O2 & were harvested for immunoblot assay for HIF-1 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. (H) HASMCs & HUVECs were exposed to 10 µM PGE1 for 4 h under 20% O2 & were harvested for immunoblot assay for HIF-2 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. I. HASMCs were exposed to 1 µM PGE1, lipo-PGE1 & PGE1-alfadex under 20% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - Effect of PGE1 on stability & synthesis of HIF-1 alpha.(A) Human aortic smooth muscle cells (HASMCs) & human umbilical vein endothelial cells (HUVECs) were exposed to 1 µM PGE1 or 100 µM DFX or incubated for 4 h, & CHX was added to a final concentration of 100 µM. The cells were incubated for 0 to 30 min, & whole-cell lysates were subjected to immunoblot assay using anti-HIF-1 alpha or -beta antibodies. (B) Serum-starved HASMCs were pretreated with no drug & 1 µM PGE1 for 30 min in Met-free medium. [35S]Met-Cys was added, & the cells were incubated for 60 min prior to preparation of cell lysates. Aliquots of 1 mg of the lysates were subjected to immunoprecipitation with anti-HIF-1 alpha antibody, separated by SDS-PAGE & exposed. Aliquots of 50 µg of the same lysate were separately subjected to immunoblotting analysis with anti-beta -actin antibody. (C) HASMCs & HUVECs were exposed to vehicle or 1 µM PGE1 for 4 h in the presence of 10 µM LY294002 (LY), 50 µM PD98059 (PD), or 5 µM GF109203X (GF). The cells were harvested & the whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha & beta-actin protein expression. Experiments were repeated at least twice. Representative immunoblots are shown. (D) HASMCs were exposed to vehicle or 1 µM PGE1 for 12 h in the presence of 10 µM LY294002 (LY), 50 µM PD98059 (PD), or 5 µM GF109203X (GF). Cells were harvested & subjected to semi-quantitative RT-PCR for VEGF & GLUT1. Experiments were repeated three times. Fold induction relative to that under 20% O2 without PGE1 treatment was plotted. ∗P < 0.05 compared with the control (20%, PGE1 treatment without any kinase inhibitors). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - Effect of PGE1 on stability & synthesis of HIF-1 alpha.(A) Human aortic smooth muscle cells (HASMCs) & human umbilical vein endothelial cells (HUVECs) were exposed to 1 µM PGE1 or 100 µM DFX or incubated for 4 h, & CHX was added to a final concentration of 100 µM. The cells were incubated for 0 to 30 min, & whole-cell lysates were subjected to immunoblot assay using anti-HIF-1 alpha or -beta antibodies. (B) Serum-starved HASMCs were pretreated with no drug & 1 µM PGE1 for 30 min in Met-free medium. [35S]Met-Cys was added, & the cells were incubated for 60 min prior to preparation of cell lysates. Aliquots of 1 mg of the lysates were subjected to immunoprecipitation with anti-HIF-1 alpha antibody, separated by SDS-PAGE & exposed. Aliquots of 50 µg of the same lysate were separately subjected to immunoblotting analysis with anti-beta -actin antibody. (C) HASMCs & HUVECs were exposed to vehicle or 1 µM PGE1 for 4 h in the presence of 10 µM LY294002 (LY), 50 µM PD98059 (PD), or 5 µM GF109203X (GF). The cells were harvested & the whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha & beta-actin protein expression. Experiments were repeated at least twice. Representative immunoblots are shown. (D) HASMCs were exposed to vehicle or 1 µM PGE1 for 12 h in the presence of 10 µM LY294002 (LY), 50 µM PD98059 (PD), or 5 µM GF109203X (GF). Cells were harvested & subjected to semi-quantitative RT-PCR for VEGF & GLUT1. Experiments were repeated three times. Fold induction relative to that under 20% O2 without PGE1 treatment was plotted. ∗P < 0.05 compared with the control (20%, PGE1 treatment without any kinase inhibitors). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - PGE1 induces HIF-1 alpha protein accumulation in vascular-derived cells.Human aortic smooth muscle cells (HASMCs) (A & B), human umbilical vein endothelial cells (HUVECs) (C & D), & HEK293 cells (G) were exposed to 1 or 10 µM PGE1 under 20% O2 or 1% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha, HIF-1 beta, & beta-actin protein expression. Experiments were repeated thrice (A, C & G). Representative immunoblots are shown (A & C). Band intensities were analyzed densitometrically (B & D). Fold induction relative to lane 1 was plotted as mean ± S.D. ∗P < 0.05 compared with the control. HASMCs (E) & HUVECs (F) were exposed to 1 µM PGE1 for the indicated times under 20% O2 & were harvested for immunoblot assay for HIF-1 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. (H) HASMCs & HUVECs were exposed to 10 µM PGE1 for 4 h under 20% O2 & were harvested for immunoblot assay for HIF-2 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. I. HASMCs were exposed to 1 µM PGE1, lipo-PGE1 & PGE1-alfadex under 20% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - Differential involvement of EP receptors in PGE1-induced HIF-1 alpha protein accumulation.(A) Expression of EP1, EP2, EP3, & EP4 receptors in human aortic smooth muscle cells (HASMCs), human umbilical vein endothelial cells (HUVECs), & cells of the neuroblastoma cell line SH-SY5Y. HASMCs, HUVECs, & SH-SY5Y cells were cultured under 20% O2 & harvested for semi-quantitative RT-PCR for EP1-4 receptors. Experiments were repeated at least three times in triplicate. Fold induction relative to expression in SH-SY5Y cells was plotted as mean ± S.D. HASMCs & HUVECs were exposed to 1 µM of EP-receptor-specific agonists (ONO-DI-004 for EP1, ONO-AE1-259-01 for EP2, ONO-AE-248 for EP3, & ONO-AE1-329 for EP4) for 4 h (B). HASMCs & HUVECs were exposed to 1 µM PGE1 with or without 1 µM EP-receptor-specific antagonists (ONO-8713 against EP1, ONO-AE3-240 against EP3, & ONO-AE2-227 against EP4) for 4 h (C). The cells were harvested & the whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha & beta-actin protein expression. Experiments were repeated twice. Representative immunoblots are shown. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - HIF was not the only factor stabilizing activated EGFR in VHL-deficient ccRCC cells.A. Western blot analysis of 786-VHL & 786-mock cells stably expressing shRNA constructs. For HIF2 alpha & HIF1 beta analysis, nuclear extracts were generated & analyzed. Anti-HA blot detected HA-VHL. B. 786-VHL cells expressing SCR (control), HIF2a-566 & HIF2a-1631 (shRNA constructs against HIF2 alpha) were treated with EGF & analyzed as described in Fig. 1A. C. 786-mock cells expressing SCR (control sequence), HIF2a-566 & HIF2a-1631 (shRNA constructs against HIF2 alpha) were treated with EGF & analyzed as described in Fig. 2B. D. The EGFR signals in Fig. 2B & 2C were normalized over Vinculin via densitometry & plotted over time. Means & SDs of three separate experiments were shown. Image collected & cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0023936), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Immunocytochemistry/ Immunofluorescence: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - Co-localization of HIF-2 alpha with HIF-1 beta & RNAPII. (a) Immunofluorescence detecting indicated proteins using antibodies labelled with Alexa Fluor 555 (red, pseudocolour) & Alexa Fluor 488 (green, pseudocolour). ‘Merge’ is the red image superimposed onto the green image of the co-stained nuclear proteins. ‘Coloc’ is the co-localization channel calculated using ImageJ plugin Co-localization Threshold. White indicates pixels where both red & green signal is found (i.e. co-localization). ‘Overlay’ is the co-localization image superimposed onto the merged image. Inlay is the magnified region (white square). White arrows highlight regions of co-localization. Scale bar, 5 μm. Abbreviations: RNAPII, RNA Polymerase II phospho-serine 5. (b) Immunofluorescence images were analysed using ImageJ plugin Co-localization Threshold with use of the Costes et al. [31] method to automatically create a threshold prior to calculating the Mander's coefficient for both proteins. The results are given as, for example, the percentage of protein A (HIF-2 alpha) that co-localized with protein B (HIF-1 beta or RNAPII) & vice versa. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27655733), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - Differential involvement of EP receptors in PGE1-induced HIF-1 alpha protein accumulation.(A) Expression of EP1, EP2, EP3, & EP4 receptors in human aortic smooth muscle cells (HASMCs), human umbilical vein endothelial cells (HUVECs), & cells of the neuroblastoma cell line SH-SY5Y. HASMCs, HUVECs, & SH-SY5Y cells were cultured under 20% O2 & harvested for semi-quantitative RT-PCR for EP1-4 receptors. Experiments were repeated at least three times in triplicate. Fold induction relative to expression in SH-SY5Y cells was plotted as mean ± S.D. HASMCs & HUVECs were exposed to 1 µM of EP-receptor-specific agonists (ONO-DI-004 for EP1, ONO-AE1-259-01 for EP2, ONO-AE-248 for EP3, & ONO-AE1-329 for EP4) for 4 h (B). HASMCs & HUVECs were exposed to 1 µM PGE1 with or without 1 µM EP-receptor-specific antagonists (ONO-8713 against EP1, ONO-AE3-240 against EP3, & ONO-AE2-227 against EP4) for 4 h (C). The cells were harvested & the whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha & beta-actin protein expression. Experiments were repeated twice. Representative immunoblots are shown. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Immunocytochemistry/ Immunofluorescence: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - Sub-nuclear localization of HIF-1 alpha & HIF-2 alpha. (a) HeLa cells ectopically expressing HIF-1 alpha & HIF-2 alpha EGFP fusions compared with endogenous HIF-1 alpha & HIF-2 alpha labelled using immunostaining. Images of HIF-1 alpha were taken following DMOG treatment (6 h; 0.5 mM). Scale bar, 5 μm. (b) HeLa cells transiently transfected with plasmids encoding (i) clover-HIF-2 alpha (green, pseudocolour), (ii) dsRED-HIF-2 alpha (red, pseudocolour), (iii) HIF-2 alpha-venus (yellow pseudocolour) & (iv) Halotag-HIF-2 alpha (green, pseudocolour). The cells expressing Halotag-HIF-2 alpha were labelled with the fluorescent Oregon Green Halotag ligand (HL-OregonGreen; Promega, WI, USA) to visualize the fusion protein. (c) Confocal images of C2C12 (mouse myoblast; top) & HEK293T (Human embryonic kidney cells; bottom) cells ectopically expressing EGFP-HIF-2 alpha. Scale bar, 5 μm. (d) HeLa cells transiently transfected with EGFP-HIF-2 alpha were imaged with a CCD camera. One thousand frames were acquired per cell in normoxia, hypoxia (1% v/v O2, 16 h) or following treatment with DMOG (0.5 mM, 6 h). The average (±s.d.) number of speckles per nucleus in each condition was 64 ± 49 (n = 25), 44 ± 24 (n = 24) & 96 ± 33 (n = 22), respectively. Mean of the sample data represented by the red dashed line. (e) Using the images from (d) the average speckle area per nucleus over the 1000 frames. The mean values (±s.d.) for each condition were 0.24 ± 0.09 µm (n = 25), 0.21 ± 0.07 µm (n = 24) & 0.27 ± 0.09 µm (n = 22), respectively. The mean values for hypoxia & DMOG were compared with the normoxic values (independent t-test, significance value set at 5%). Mean of the sample data represented by the red dashed line. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27655733), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - PGE1 induces HIF-1 alpha protein accumulation in vascular-derived cells.Human aortic smooth muscle cells (HASMCs) (A & B), human umbilical vein endothelial cells (HUVECs) (C & D), & HEK293 cells (G) were exposed to 1 or 10 µM PGE1 under 20% O2 or 1% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha, HIF-1 beta, & beta-actin protein expression. Experiments were repeated thrice (A, C & G). Representative immunoblots are shown (A & C). Band intensities were analyzed densitometrically (B & D). Fold induction relative to lane 1 was plotted as mean ± S.D. ∗P < 0.05 compared with the control. HASMCs (E) & HUVECs (F) were exposed to 1 µM PGE1 for the indicated times under 20% O2 & were harvested for immunoblot assay for HIF-1 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. (H) HASMCs & HUVECs were exposed to 10 µM PGE1 for 4 h under 20% O2 & were harvested for immunoblot assay for HIF-2 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. I. HASMCs were exposed to 1 µM PGE1, lipo-PGE1 & PGE1-alfadex under 20% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - PGE1 induces HIF-1 alpha protein accumulation in vascular-derived cells.Human aortic smooth muscle cells (HASMCs) (A & B), human umbilical vein endothelial cells (HUVECs) (C & D), & HEK293 cells (G) were exposed to 1 or 10 µM PGE1 under 20% O2 or 1% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha, HIF-1 beta, & beta-actin protein expression. Experiments were repeated thrice (A, C & G). Representative immunoblots are shown (A & C). Band intensities were analyzed densitometrically (B & D). Fold induction relative to lane 1 was plotted as mean ± S.D. ∗P < 0.05 compared with the control. HASMCs (E) & HUVECs (F) were exposed to 1 µM PGE1 for the indicated times under 20% O2 & were harvested for immunoblot assay for HIF-1 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. (H) HASMCs & HUVECs were exposed to 10 µM PGE1 for 4 h under 20% O2 & were harvested for immunoblot assay for HIF-2 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. I. HASMCs were exposed to 1 µM PGE1, lipo-PGE1 & PGE1-alfadex under 20% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] -

Western Blot: ARNT/HIF-1 beta Antibody (H1beta234) - BSA Free [NB100-124] - PGE1 induces HIF-1 alpha protein accumulation in vascular-derived cells.Human aortic smooth muscle cells (HASMCs) (A & B), human umbilical vein endothelial cells (HUVECs) (C & D), & HEK293 cells (G) were exposed to 1 or 10 µM PGE1 under 20% O2 or 1% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha, HIF-1 beta, & beta-actin protein expression. Experiments were repeated thrice (A, C & G). Representative immunoblots are shown (A & C). Band intensities were analyzed densitometrically (B & D). Fold induction relative to lane 1 was plotted as mean ± S.D. ∗P < 0.05 compared with the control. HASMCs (E) & HUVECs (F) were exposed to 1 µM PGE1 for the indicated times under 20% O2 & were harvested for immunoblot assay for HIF-1 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. (H) HASMCs & HUVECs were exposed to 10 µM PGE1 for 4 h under 20% O2 & were harvested for immunoblot assay for HIF-2 alpha protein. Experiments were repeated twice. Representative immunoblots are shown. I. HASMCs were exposed to 1 µM PGE1, lipo-PGE1 & PGE1-alfadex under 20% O2 conditions for 4 h. After treatment, cells were harvested & whole-cell lysates were subjected to immunoblot assay for HIF-1 alpha. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24349900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.