Anti-HIF-1-alpha/HIF1A Antibody Picoband®

Western blot analysis of HIF 1 alpha using anti-HIF 1 alpha antibody (PB9253).
Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: Untreated human HepG2 whole cell lysates,
Lane 2: Cobalt Chloride treated human HepG2 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-HIF 1 alpha antigen affinity purified polyclonal antibody (PB9253) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054) at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for HIF 1 alpha at approximately 120 kDa. The expected band size for HIF 1 alpha is at 93 kDa.

Western blot analysis of HIF1A using anti-HIF1A antibody (PB9253).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: mouse 4T1 whole cell lysates,
Lane 2: LPS-stimulated mouse 4T1 whole cell lysates,
Lane 3: Low-dose drug mouse 4T1 whole cell lysates,
Lane 4: Medium-dose drug mouse 4T1 whole cell lysates,
Lane 5: High-dose drug mouse 4T1 whole cell lysates,
Lane 6: Positive control drug mouse 4T1 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1 hour at RT. The membrane was incubated with rabbit anti-HIF1A antigen affinity purified monoclonal antibody (Catalog # PB9253) at 1:2500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with ChemiDoc MP system. A specific band was detected for HIF1A at approximately 110-120 kDa. The expected band size for HIF1A is at 120kDa.

IHC analysis of HIF 1 alpha using anti-HIF 1 alpha antibody (PB9253).
HIF 1 alpha was detected in paraffin-embedded section of mouse intestine tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HIF 1 alpha Antibody (PB9253) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of HIF 1 alpha using anti-HIF 1 alpha antibody (PB9253).
HIF 1 alpha was detected in paraffin-embedded section of rat intestine tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HIF 1 alpha Antibody (PB9253) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of HIF 1 alpha using anti-HIF 1 alpha antibody (PB9253).
HIF 1 alpha was detected in paraffin-embedded section of human intestinal cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HIF 1 alpha Antibody (PB9253) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

HIF-1α expression was analyzed by immunohistochemistry. Results of a 61-year-old male patient who underwent NAC and surgery treatment for PDAC, and had a CAP score of 2, which indicated that he was a responder. HIF-1α was highly expressed in the (A) nucleus (weighted score 12), (B) cytoplasm (weighted score 12), and (C) stroma (weighted score 4); results of a 30-year-old male patient who underwent NAC and surgery treatment for PDAC, and had a CAP score of 3, which indicated that he was a non-responder. HIF-1α was lowly expressed in the (D) nucleus (weighted score 4), (E) cytoplasm (weighted score 4), and (F) stroma (weighted score 4). 40×; scale bar =20 µm. NAC, neoadjuvant chemotherapy; PDAC, pancreatic ductal adenocarcinoma; CAP, the College of American Pathologists; HIF-1α, hypoxia-inducible factor-1-alpha.
Index in PubMed under a CC BY license. PMID: 39839013

Histological and immunohistochemical images of the rat livers of the control group and hypoxia group of CLD. (A) H&E staining images (× 40) of the control and hypoxia group of CLD. (B) Masson staining images (× 40) of the control group and hypoxia group of CLD. (C) Pimonidazole Immunohistochemical staining images (× 40) of control and hypoxia group of CLD. (D) HIF-1α Immunohistochemical staining images (× 40) of control and hypoxia group of CLD. (E) α-SMA Immunohistochemical staining images (× 40) of control and hypoxia group of CLD.
Index in PubMed under a CC BY license. PMID: 39906347

Tumors from CD47-deficient mice exhibited improved tumor angiogenesis and vascular integrity compared to those from WT mice. Tumors were surgically removed from WT or CD47 −/− mice 11 days after tumor cell injection for the following analyses ( n = 4 per group). ( A ) Representative images of CD31 staining (black arrow) of tumor sections (Scale bar, 20 μm). ( B ) The microvessel density (MVD) quantified by counting positive cells in six randomly selected fields (400×) using Image Pro Plus 6.0 software. ( C ) CD31 mRNA levels in tumors quantified by real-time qPCR. ( D ) Representative images of HIF-1A staining (pink) of tumor sections (Scale bar, 20 μm). ( E ) Percentages of HIF-1A + cells in tumors quantified by counting positive cells in six randomly selected fields (400×) using Image Pro Plus 6.0 software. ( F ) Representative images of CD144 staining (red) of tumor sections (Scale bar, 20 μm). ( G ) Percentages of CD144 + area in tumors quantified using Image Pro Plus 6.0 software. Data are mean ± SDs. ** P < 0.01, *** P < 0.001.
Index in PubMed under a CC BY license. PMID: 27283989

AhR transcriptionally activates miR-142a to inhibit IRF1 and HIF-1α expression. (A) The top 30 miRNAs in BMDMs that are regulated by LPS are arranged in a miRNA array heatmap. In addition, miRNAs predicted to be under the transcriptional control of AhR (according to analysis with the JASPAR database) are noted. (B) Venn diagram analyses were performed to identify miRNAs that can both target IRF1 and HIF-1α and that are under the transcriptional control of AhR. (C) Renal expression of mmu-miR-142a-3p in mice (n = 6) with CaOx nephrocalcinosis following treatment with an AhR neutralizing antibody or FICZ treatment was assessed via FISH (200×; scale bar: 20 µm). (D) qRT-PCR was performed to measure mmu-miR-142a-3p expression in BMDMs using U6 RNA as a normalization control. (E, F) ChIP assays and ChIP qPCR analysis showed that AhR bound to the miR-142a promoter in BMDMs treated with the AhR overexpression plasmid. (G) A schematic model showed that AhR directly binds to the miR-142a promoter and activates its transcription. (H, I) WT and mutated miR-142a targeting sequences in the IRF1 and HIF-1α 3'-UTR regions that were used to construct luciferase reporters, with reporters bearing these IRF1 (J) or HIF-1α (L) 3'-UTR sequences co-transfected along with miR-142a mimic (100 nM). IRF1 (K) and HIF-1α (M) mRNA levels were detected via qRT-PCR in BMDMs following miR-142a mimic or inhibitor transfection. Western blotting (N, O) analysis enabled the detection of IRF1 and HIF-1α expression while also assessing the levels of iNOS and Arg-1 to monitor the polarization state of BMDMs following miR-142a mimic or inhibitor transfection. β-actin was employed as a normalization control. The data are shown as the means ± SD of triplicate experiments. *P < 0.05; **P < 0.01, as assessed via Student's t test (D, F) or one-way ANOVA (J-M, O).
Index in PubMed under a CC BY license. PMID: 33204326

AhR activation in vitro decrease M1 macrophage polarization to inhibit kidney inflammation and injury through the AhR-miR-142a-IRF1/HIF-1α axis in vitro . (A) Western blotting analysis enabled the detection of AhR, HIF-1α, IRF1, NF-κB p65, iNOS, and Arg-1 expression in BMDMs. β-actin was detected as an internal control. (B) iNOS (M1 macrophage marker, green) and Arg-1 (M2 macrophage marker, red) distributions in BMDMs were detected by immunofluorescence (200×; scale bar: 20 µm). (C) Schematic diagram of BMDMs phagocytic capacity testing. (D) Fluorescence microscopy was performed to analyse the phagocytic ability of BMDMs (200×; scale bar: 20 µm). (E) qRT-PCR analysis of iNOS, IL-6, CIITA, Arg-1, Chi3l3 and Fizz1 expression to further determine polarization state of BMDM. (F) ELISA was used to quantify cytokine levels in the co-culture media. The data are shown as the means ± SD of triplicate experiments. *P < 0.05; **P < 0.01, as assessed via one-way ANOVA (E, F).
Index in PubMed under a CC BY license. PMID: 33204326

Construction of animal models and analysis of pathological damage. (A) TTC staining plots of the Sham and VD group. (B) Motion trajectory diagram. (C) Representative images stained with HE (scale bars = 50 μm). (D) Representative histopathological images obtained from TUNEL staining (scale bars = 50 μm), showing changes in the cortex hippocampus CA1. (E) Quantification of TUNEL+ cells in the hippocampal CA1 (n = 3). (F) Expression profile of CD31 between Sham and VD groups (qPCR). **P < 0.01. (G) Expression profile of HIF-1α between Sham and VD groups (qPCR; n = 6). ***P < 0.001. (H) WB strips. (I) Expression profile of CD31 between Sham and VD groups (WB). *P < 0.05. (J) Expression profile of HIF-1α between Sham and VD groups (WB; n = 3). ***P < 0.001.
Index in PubMed under a CC BY license. PMID: 40988927

Receiver operator characteristic curves of nuclear HIF-1α expression (A), %ΔCA19-9 (B), and tumor differentiation (C) for predicting the post-NAC pathological response. HIF-1α, hypoxia-inducible factor-1α; NAC, neoadjuvant chemotherapy; %Δ, [(post-NAC − pre-NAC)/pre-NAC]; CA19-9, carbohydrate antigen 19-9; AUC, area under the curve; CI, confidence interval.
Index in PubMed under a CC BY license. PMID: 39839013

Effect of neohesperidin and naringin on cell viability, HIF-1α, Caspase3, PLIN2 in PASMCs. A – D Effects of neohesperidin and naringin on PASMCs viability were observed by MTT (n = 6). E – J Expression levels of HIF-1α, Caspase3, PLIN2 in PASMCs were evaluated by Western Blot (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs control, # p < 0.05, ## p < 0.01, ### p < 0.001 vs PDGF-BB. 5 represents 5 µM, 10 represents 10 µM
Index in PubMed under a CC BY license. PMID: 39696593

ZXGD modulated HIF-1α mediated pulmonary vascular remodeling. A Expression level of HIF-1α in lung tissue of rats was tested with PCR (n = 3). B Expression level of HIF-1α in PASMCs transfected with siRNA (n = 6). C Effects of ZXGD on viability of PASMCs transfected with HIF-1α siRNA was examined by MTT (n = 6). D – G Levels of IL-6, IL-10, LDL-C and decadienyl- l -carnitine in PASMCs transfected with HIF-1α siRNA. H – K Expression level of HIF-1α, Caspase-3, PCNA, PLIN2 in PASMCs transfected with HIF-1α siRNA (n ≥ 3). * p < 0.05, *** p < 0.001 vs control, # p < 0.05, ## p < 0.01, ### p < 0.001 vs PH or PDGF-BB
Index in PubMed under a CC BY license. PMID: 39696593

ZXGD inhibited hypoxia, oxidative stress and inflammation in rats with PH and PASMCs induced with PDGF-BB. A – E Expression of HIF-1α, Nrf2, IL-1β, IL-6 and IL-10 in lung tissue of rats were detected using Western blot (n = 3 or 6). F – I Concentration of IL-6, IL-1β, TNF-α and IL-10 in lung tissue of rats were examined by ELISA (n = 6). J Level of ROS in PASMCs was assessed with flow cytometry, and statistical data was obtained. ** p < 0.01, *** p < 0.001 vs control, # p < 0.05, ## p < 0.01, ### p < 0.001 vs PH or PDGF-BB
Index in PubMed under a CC BY license. PMID: 39696593

Correlations for the hypoxia score (HIF-1α) with the pseudo-diffusion coefficient (D*) and the T1 mapping. (A) HIF-1α correlated positively with the D* ( r = 0.556, p = 0.020). (B) HIF-1α correlated positively with T1 mapping ( r = 0.505, p = 0.039).
Index in PubMed under a CC BY license. PMID: 39906347

Effects of XLW on HIF1α and PTGS2 expressions in rat extraocular muscle tissues. ( A ) The immunohistochemical slice of HIF1α, ( B ) HIF1α immunohistochemical quantitative analysis, ( C ) WB of PTGS2 levels, and ( D ) quantification of the WB in PTGS2. ** p < 0.01, **** p < 0.0001 versus NC group; # p < 0.05, ## p < 0.01 versus HT group; n = 6 or 8
Index in PubMed under a CC BY license. PMID: 40696458

Response of ZF4 cells to hypoxic stress. (A) Cell Viability was analyzed with PrestoBlue™ HS Cell Viability Regent under normoxia or hypoxia treated for 1, 2, 3 and 4 d. (B) The mRNA expression of iron absorption and storage gene in ZFL cells was quantified by real-time RT–PCR under normoxia and hypoxia for 3 days. (C) Western blot analysis of Hif-1α and Ferritin expression in ZF4 cells cultured under normoxia and hypoxia for 3 days. (D) The microscope analysis of morphology changes in ZF4 cells under normoxia or hypoxia treated with or without FAC (2.5 mM), DFO (10 µM) and Fer-1 (2.5 µM) for 3 days. (E) Cell Viability was analyzed with PrestoBlue™ HS Cell Viability Regent under normoxia or hypoxia treated with or without FAC (2.5 mM), DFO (10 µM) and Fer-1 (2.5 µM). (F–H) Analysis of changes in general ROS (F) , mitochondrial-derived ROS (G) and lipid peroxidation (H) levels in cells with H2DCFDA, MitoSOX and C11-BODIPY probe under normoxia and hypoxia for 3 days. Cells under hypoxic stress were rescued with FAC (2.5 mM), DFO (10 µM) and Fer-1 (2.5 µM). Normoxia was used as a control group for significance analysis. Error bars, mean ± s.d., n = 3 (biological replicates).
Index in PubMed under a CC BY license. PMID: 36091397

AhR significantly suppressed IRF1 and HIF-1α expression in a murine CaOx nephrocalcinosis model. (A) RNA-seq heatmap showing significantly altered mRNAs in SGA-treated BMDMs. (B) Volcano plots showing mRNA transcripts that were differentially expressed between LPS-treated and SGA-treated BMDMs. Significantly downregulated and upregulated mRNAs are shown in green and red, respectively, whereas genes that were not significantly changed are shown in black. (C) IHC staining for AhR, IRF1, and HIF-1α in the kidneys of FICZ-treated mice with CaOx nephrocalcinosis (200×; scale bar: 20 µm). (D) qRT-PCR was used to assess AhR, IRF1, and HIF-1α expression in kidney samples from FICZ-treated mice (n = 6) with CaOx nephrocalcinosis compared to kidney samples from model mice. (E, F) Pearson's correlation coefficient analysis (n = 30) of the expression levels of AhR and IRF1 (E) or HIF-1α (F). Each dot represents an individual animal. *P < 0.05; **P < 0.01, as assessed via one-way ANOVA (D).
Index in PubMed under a CC BY license. PMID: 33204326

Comparison of CT values between the nuclear HIF-1α-high and -low expression PDAC patients following NAC. ***, P<0.001; **, P=0.003. CT, computed tomography; HIF-1α, hypoxia-inducible factor-1-alpha; PDAC, pancreatic ductal adenocarcinoma; NAC, neoadjuvant chemotherapy; AP, arterial phase; DP, delayed phase; HU, c.
Index in PubMed under a CC BY license. PMID: 39839013

AhR suppressed IRF1 and HIF-1α to attenuate CaOx crystal-stimulated M1 macrophage polarization in vitro . (A) BMDMs and COM-treated TECs co-culture model. (B, C) Western blotting analysis was used to detect AhR, HIF-1α, IRF1, NF-κB p65, iNOS, and Arg-1 expression after FICZ treatment and the upregulation or downregulation of AhR in BMDMs. β-actin served as a normalization control. (D, E) iNOS (M1 macrophage marker, green) and Arg-1 (M2 macrophage marker, red) distribution in BMDMs were detected by immunofluorescence (200×; scale bar: 20 µm). (F, G) qRT-PCR analysis of iNOS, IL-6, CIITA, Arg-1, Chi3l3 and Fizz1 expression to further determine polarization state of BMDM. The data are shown as the means ± SD of triplicate experiments. *P < 0.05; **P < 0.01, as assessed via one-way ANOVA (F, G).
Index in PubMed under a CC BY license. PMID: 33204326

AhR activation suppressed the deposition of CaOx crystal and CaOx nephrocalcinosis-mediated kidney inflammation and injury through the AhR-miR-142a-IRF1/HIF-1α axis in vivo . (A) Experimental overview. (B) The deposition of renal CaOx crystal in FICZ- and/or antagomiR-142a-treated mice was assessed via polarized light optical microscopy (20×; scale bar: 500 µm). Pizzolato staining was employed as a means of detecting these CaOx crystal in corticomedullary tissue, while PAS was utilized to evaluate injury to TECs (200×; scale bar: 20 µm), and TUNEL staining was employed to assess renal TECs death (200×; scale bar: 50 µm). (C) PET-CT scanning was employed as a means of assessing renal inflammation state in CaOx nephrocalcinosis mice. (D) IHC was used to analyse AhR, IRF1, and HIF-1α expression, and FISH was used to detect miR-142a expression in renal tissue (200×; scale bar: 20 µm). (E) iNOS (M1 macrophage marker, red) and Arg-1 (M2 macrophage marker, green) distributions in renal tissues were detected by immunofluorescence (200×; scale bar: 50 µm). (F) On days 3 and 10, the serum pro-inflammatory IL-1β, TNF-α, and IL-6 levels and the anti-inflammatory IL-10 levels were measured by ELISA. n = 6 per group. *P < 0.05; **P < 0.01, as assessed via one-way ANOVA (F).
Index in PubMed under a CC BY license. PMID: 33204326