rabbit anti hif2a  (Novus Biologicals)

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a , GO enrichment analysis was conducted to elucidate the molecular functions (MF), biological processes (BP), and cellular components (CC) associated with proteins predicted by the HuRI to potentially interact with BMAL1; two-sided Fisher’s exact test, Benjamini–Hochberg correction applied. b , Western Blot analysis of reciprocal co-IP with HIF2A in hypoxia-treated (1% O 2 , 4 h) or normoxia-treated HEK293 cells. Cytosolic and nuclear protein extracts were immunoprecipitated with HIF2A and blotted with anti-BMAL1, anti-HIF2A, anti-HIF1B, anti-β-actin, and anti-Lamin A/C antibodies. An IgG control affirmed procedure specificity. H indicates HIF2A. n = 3 independent experiments. c , Size-exclusion chromatography analysis of the BMAL1/HIF2A heterodimer. The purified BMAL1/HIF2A complex was loaded onto a Superdex 200 Increase 10/300 GL column. n = 3 independent experiments. The molecular weights of makers are as indicated. d , SDS-PAGE analysis of the recombinant BMAL1/HIF2A heterodimer purified by size-exclusion chromatography. n = 3 independent experiments.

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Size-exclusion Chromatography, Purification, SDS Page, Recombinant

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a , Predicted interactions of BMAL1 with bHLH-PAS transcription factors in the human LV using the HuRI. b , c , Co-IP analysis ( b ) and quantification ( c ) of BMAL1–Flag in HEK293 cells. n = 3 independent experiments. Cyto., cytoplasmic fraction; nuc., nuclear fraction. d , Co-IP analysis of HIF2A in HCMs. n = 3 independent experiments. e , f , PLA ( e ) and quantification ( f ) showing BMAL1–HIF2A interactions in HCMs. White arrows indicate the close interaction between BMAL1–HIF2A or BMAL1–HIF1A in the nuclei. Scale bar, 25 μm. n = 20 (BMAL1–HIF2A and BMAL1–HIF1A) and n = 10 (negative control). g , Schematic of BMAL1 and HIF2A protein domains. h , Flag pull-down of BMAL1. n = 3 independent experiments. i , j , Western blot analysis ( i ) and quantification ( j ) of nuclear BMAL1 and HIF2A levels in the AAR of C57BL/6J mice after 2 h reperfusion at ZT8 or ZT20. n = 3 mice per group per timepoint. Owing to similar molecular masses of proteins, the samples were run on separate gels, with TBP as the sample processing control. k , Immunofluorescence analysis of BMAL1 and HIF2A colocalization in the border zone on day 1 after MI, shown in merged images (yellow, white arrows). Scale bars, 25 μm. n = 3 mice per group per timepoint. l – o , Hif2a loxP/loxP myosin-Cre + mice and myosin-Cre + mice were subjected to IRI at ZT8 or ZT20. Evan’s blue and TTC-stained heart slices ( l ; scale bar, 1 mm), AAR as the percentage of LV ( m ), infarct size as the percentage of the AAR ( n ) and serum troponin I levels ( o ) after 2 h reperfusion are shown. n = 8 mice per group per timepoint. p – s , Cardiac function by day 14 after MI. p , The EF and FS. q , The GLS. r , LV 3D and six-segment longitudinal strain images. s , Intraventricular dyssynchrony. For p – s , n = 8 ( Hif2a loxP/loxP myosin-Cre + ) and n = 9 (myosin-Cre + ) mice per timepoint. Data are mean ± s.e.m. Statistical analysis was performed using one-way ANOVA ( c ) and two-way ANOVA ( f , j , m – q and s ).

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: Co-Immunoprecipitation Assay, Negative Control, Western Blot, Control, Immunofluorescence, Staining

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a , Representative trichrome staining of heart sections on day 1 post‐MI. Border and remote areas are identified with an arrow and a star, respectively. The dashed lines highlight the extension of the areas of ischaemic injury, scale bar, 1 mm. b and d , Representative immunostaining of HIF2A (red) (b) , BMAL1 (red) (d) , WGA (green), and nuclei (DAPI; blue) in the border zone and remote area of hearts from C57BL/6J mice subjected to myocardial IRI at ZT8 or ZT20, n = 4 mice/group/time point, scale bar, 20 μm. White arrows indicate HIF2A or BMAL1 within the nuclei and cytoplasm. c and e , Quantification of fluorescence intensity of HIF2A in (b) and BMAL1 in (d) . Normalized to the protein levels in the remote areas at ZT8. Each quantification value dot represents the average value of three fields in one section. n = same as above. Data are mean ± s.e.m. Statistical analysis was performed using two-way ANOVA.

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: Staining, Immunostaining, Fluorescence

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a , Heat map of the top 20 potential HIF2A targets. n = 4 mice per group. b – d , HIF2A targets ( b ) and AREG protein (western blot ( c ) and quantification ( d )) in the AAR of C57BL/6J mice after 2 h reperfusion. n = 3 mice per group per timepoint. Owing to similar molecular masses of proteins, the samples were run on separate gels, with α-tubulin as the sample processing control. e , f , AREG immunostaining in the border zone on day 1 after MI ( e ; scale bars, 25 μm) and quantification ( f ). Arrows indicate α-sarcomeric + /AREG + cells. n = 4 mice per timepoint. g – i , Synchronized HCMs were exposed to hypoxia (1% O 2 , 4 h) across circadian times (CT0–CT40); AREG mRNA expression ( g ) and BMAL1–HIF2A/AREG protein ( h ) and quantification ( i ) are shown. n = 3. j , Analysis of AREG mRNA in HEK293 cells transfected with various siRNAs and exposed to hypoxia. n = 3. k – m , The fold change in Areg mRNA transcripts ( k ), and western blot ( l ) and quantification ( m ) of cardiomyocyte AREG after 2 h reperfusion in the AAR of myosin-Cre + , Bmal1 loxP/loxP myosin-Cre + and Hif2a loxP/loxP myosin-Cre + mice. n = 5 (transcripts) mice per group; and n = 5, 5 and 4 (proteins) mice. Statistical analysis was performed using unpaired two-tailed t -tests, with Welch’s t -tests comparing between myosin-Cre + and Hif2a loxP/loxP myosin-Cre + mice (protein). n , o , Surface plasmon resonance analysis of BMAL1–HIF2A binding to HRE ( n ) or E-box ( o ). n = 3. p , Conserved BMAL1–HIF2A-binding site (CAGGTG) on the AREG promoter. q , ChIP–qPCR analysis of HIF2A binding in HEK293 cells at this shared site at CT20 or CT32. n = 5. r , Co-IP analysis of HIF2A–BMAL1 interactions in HCMs at CT20/CT32. n = 3. s , ChIP–qPCR analysis of HEK293 cells for HIF2A ( n = 13) and BMAL1 (n = 12 (normoxia) and n = 11 (hypoxia)). t , Luciferase assays of h AREG promoter activation in HEK293 cells. n = 4. All of the samples are biologically independent. Data are mean ± s.e.m. Statistical analysis was performed using one-way ANOVA ( g , i , j and t ), two-way ANOVA ( b , d , q ), unpaired two-tailed t -tests ( f ) and two‐sided Mann–Whitney U -test ( s ).

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: Western Blot, Control, Immunostaining, Expressing, Transfection, Two Tailed Test, SPR Assay, Binding Assay, ChIP-qPCR, Co-Immunoprecipitation Assay, Luciferase, Activation Assay, MANN-WHITNEY

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a-d , C57BL/6J mice treated with vadadustat (vada, 50 mg/kg, i.p., daily) or Vehicle (Veh) for three days at ZT8 or ZT20. HIF2A and HIF1A protein levels assessed by Western blot ( a ) and quantified ( b ). n = 3 mice/group/time point; two-way ANOVA. Immunostaining of HIF2A (red), WGA (green), and nuclei (DAPI, blue) in mouse hearts ( c ; scale bar: 25 μm), with quantification of HIF2A fluorescence intensity ( d ). n = 4 mice/Veh-treated/ZT8, n = 4 mice/Veh-treated/ZT20, n = 3 mice/vada-treated/ZT8, and n = 3 mice/vada-treated/ZT20; two-way ANOVA. e-g , Mice given an additional dose of vadadustat 2 h before IRI at ZT8 or ZT20. Evan’s blue and TTC-stained heart slices ( e ), quantification of AAR as % LV ( f ), and infarct size as % AAR ( g ) after 2 h of reperfusion. n = 8 Veh-treated/ZT8, n = 8 Veh-treated/ZT20, n = 7 vada-treated/ZT8, and n = 7 vada-treated/ZT20; two-way ANOVA. h-n , Cardiac function on day 14 post-MI assessed by STE. LV systolic function (EF, FS, GLS; h ), ESV ( i ), EDLVM ( j ), B-mode imaging with 2D longitudinal strain ( k ), segmental wall contractility ( l ), 3D longitudinal strain with images showing LV motion abnormalities ( m ), and mechanical dyssynchrony measured by intra-ventricular delay ( n ). Symbols in ( m ) indicate reduced contractility (stars), dyskinesis (triangles), and dyssynchrony (circles). n = 7 mice/Veh-treated/ZT8, n = 7 mice/Veh-treated/ZT20, n = 8 mice/vada-treated/ZT8, and n = 7 mice/vada-treated/ZT20; two-way ANOVA. o and p , Mice treated with vadadustat for three days at ZT8 or ZT20, followed by IRI. Nuclear fractions from the AAR 3 h post-reperfusion were immunoprecipitated with HIF2A and blotted for HIF2A, BMAL1, and Lamin B ( o ) with quantification of BMAL1 and HIF2A protein levels ( p ). n = 3 mice/group; unpaired two-tailed t -tests. q and r , Immunostaining of AREG (red), α-sarcomeric (green), and nuclei (DAPI: blue) 3 h post-reperfusion ( q , scale bar, 25 μm), with quantification of fluorescence intensity ( r ). n = 3 mice/group; unpaired two-tailed t -tests. All data are mean ± s.e.m.

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: Western Blot, Immunostaining, Fluorescence, Staining, Imaging, Immunoprecipitation, Two Tailed Test

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a and b , HIF2A transcript levels from RNA-seq of LV biopsy samples after aortic cross-clamping in morning and afternoon cardiac surgery patients ( a , n = 56 morning, n = 17 afternoon; boxplots show the 25th and 75th percentiles (box), median (central line), and minimum to maximum values (whiskers)) and in the AAR of mice after 2 h of reperfusion at ZT8 or ZT20 ( b , n = 3 mice/time point; unpaired two-tailed t -tests). c , Hif2a transcript levels by real-time PCR after 2 h of reperfusion in the AAR subjected to IRI at ZT8 or ZT20. n = 3 mice/time point; unpaired two-tailed t -tests. d-f , Hif2a transcript levels by real-time PCR in the AAR after 2 h of reperfusion in Bmal1 loxP/loxP Myosin Cre+ mice and Myosin Cre+ mice ( d , n = 5/Myosin Cre+ and n = 3/ Bmal1 loxP/loxP Myosin Cre + ; unpaired two-tailed t -tests). HIF2A protein levels by Western blot in the nuclear fractions ( e , n = 3/Myosin Cre+ and n = 5/ Bmal1 loxP/loxP Myosin Cre + ) and quantification ( f , Welch’s t -tests). g-k , C57BL/6J mice treated with NOB (200 mg/kg, i.p.) and HIF2A protein levels were assessed by Western blot ( g, h , n = 3 mice/group/time point; two-way ANOVA) or transcript levels by real-time PCR ( i , n = 4 mice/Veh-treated/ZT8, n = 4 mice/Veh-treated/ZT20, n = 4 mice/NOB-treated/ZT8, and n = 3 mice/NOB-treated/ZT20; two-way ANOVA). Immunostaining of HIF2A in the myocardium ( j , n = 4 mice/group/time point, scale bar, 25 μm) and quantification ( k , two-way ANOVA). l - q , HCMs transduced with Bmal1-AAV or shBmal1-AAV were treated with 1% O 2 for 4 h. BMAL1 and HIF2A transcript levels by real-time PCR ( l, o ), protein levels by Western blot ( m, p ), and quantification ( n, q ). n = 3 independent experiments; Unpaired two-tailed t -tests were used for all comparisons, except for BMAL1 protein levels in ( n ), which were analysed using Welch’s t -tests. The samples used in (m ) were also used in Extended Data Fig. , sharing the β-actin blot. The samples used in ( p ) were also used in Extended Data Fig. , sharing the β-actin blot. r , ChIP-qPCR of BMAL1 binding to E-box elements in the human HIF2A promoter under normoxic and hypoxic conditions. n = 4 independent experiments; one-way ANOVA. s - v , HCMs transduced with Bmal1-AAV ( s, t ) or shBmal1-AAV ( u, v ) were exposed to 1% O 2 for 4 h and treated with CHX to assess HIF2A degradation. Protein levels were quantified and plotted to determine half-life using exponential decay. n = 3 independent experiments. w, x , HEK293 cells transfected with HIF2A-Myc, Ub-HA, and BMAL1-Flag, treated with MG132, and subjected to HIF2A immunoprecipitation. Ubiquitination levels were analysed ( w ) and quantified ( x ). n = 3 independent experiments; one-way ANOVA. All data are mean ± s.e.m.

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: RNA Sequencing, Two Tailed Test, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining, Transduction, ChIP-qPCR, Binding Assay, Transfection, Immunoprecipitation, Ubiquitin Proteomics

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a and b , RNA-seq analysis of HIF1B transcript levels in LV biopsies after aortic cross-clamping in morning vs. afternoon patients ( a , n = 56 AM, n = 17 PM; boxplots show the 25th and 75th percentiles (box), median (central line), and minimum to maximum values (whiskers)) and in the AAR after 2 h of reperfusion at ZT8 or ZT20 in mice ( b , n = 3 mice/time point; unpaired two-tailed t -tests). c-e , Hif1b transcript levels in the AAR on day 1 post-IRI in Bmal1 loxP/loxP Myosin Cre+ and Myosin Cre+ mice ( c , n = 5 Myosin Cre+ and n = 3 Bmal1 loxP/loxP Myosin Cre+ mice; unpaired two-tailed t -tests), HIF1B protein levels by Western blot ( d , n = 4 mice/group) and quantification ( e , unpaired two-tailed t -tests). f-h , Hif1b transcript levels in mouse hearts 28 days post-Bmal1-AAV or control AAV injection ( f , n = 3 mice/group; unpaired two-tailed t -tests), HIF1B protein levels by Western blot ( g , n = 3 mice/group) and quantification ( h , unpaired two-tailed t -tests). The same samples in ( g ) were also used in Supplementary Fig. , sharing the β-actin blot. i-k , HIF1B transcript levels in HCMs transduced with Bmal1-AAV or control AAV under hypoxia ( i , n = 3 independent experiments; unpaired two-tailed t -tests), HIF1B protein levels by Western blot ( j , n = 3 independent experiments), and quantification ( k ; unpaired two-tailed t -tests). The same samples used in ( j ) were also used in Extended Data Fig. , sharing the β-actin blot. l-n , HIF1B transcript levels in HCMs transduced with shBmal1-AAV or shControl AAV ( l , n = 3 independent experiments; unpaired two-tailed t -tests), HIF1B protein levels by Western blot ( m , n = 3 independent experiments), and protein quantification ( n ; unpaired two-tailed t -tests). The same samples in ( m ) were also used in Extended Data Fig. , sharing the β-actin blot. o and p , Immunoprecipitation using HIF1B and HIF2A antibodies in HEK293 cells transfected with BMAL1-Flag or control plasmid under hypoxia or normoxia, analysed by Western blot ( o , n = 3 independent experiments) and normalized to α-tubulin ( p , one-way ANOVA). q and r , ChIP-qPCR using HIF1B antibody showing binding to the human EPO ( q ) and AREG ( r ) promoters under normoxia and hypoxia. n = 8 independent experiments; two-way ANOVA. s , Luciferase assays in HEK293 cells transfected with BMAL1-Flag (0–200 nM), HIF2A-HA, and a luciferase reporter for the human PGK1 promoter. n = 4 independent experiments; one-way ANOVA. All data are mean ± s.e.m.

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: RNA Sequencing, Two Tailed Test, Western Blot, Control, Injection, Transduction, Immunoprecipitation, Transfection, Plasmid Preparation, ChIP-qPCR, Binding Assay, Luciferase

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a , A cryo-EM micrograph of the BMAL1/HIF2A/DNA complex. b , Representative 2D class averages of the BMAL1/HIF2A/DNA complex. c , Flowchart of image processing of the BMAL1/HIF2A/DNA cryo-EM data. d , FSC curves of the BMAL1/HIF2A/DNA complex. The resolution for the BMAL1/HIF2A/DNA complex is 3.6 Å according to the gold-standard FSC = 0.143 criterion. e , Cryo-EM density map of the BMAL1/HIF2A/DNA complex coloured by local resolution. f , Angular distribution plot. g , Density map of the BMAL1/HIF2A/DNA complex generated by deepEMhancer. h , Histogram and directional FSC plot generated by 3DFSC indicates that 90% of directions achieve a resolution better than 4 Å. i and j , Local structures with their corresponding densities are shown.

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: Cryo-EM Sample Prep, Generated

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a , Cryo-EM map of the BMAL1–HIF2A–DNA complex. The map was sharpened using DeepEMhancer. HIF2A and BMAL1 are coloured in purple and red, respectively, with two HRE DNA strands in green and yellow. b , The overall structure of the BMAL1–HIF2A–DNA complex. c , Individual structures of HIF2A and BMAL1 within the complex. d , The four major interfaces (I to IV) between HIF2A and BMAL1. Interaction residues in BMAL1 (red) and HIF2A (purple) are shown; the BMAL1 residues mutated for pull-down analysis in e are shown in magenta. e , Pull-down analysis showing impaired interaction between GST–HIF2A and Flag-tagged BMAL1 mutants. Mutations in the bHLH, PAS-A and PAS-B domains of the BMAL1 are indicated. n = 3 independent experiments. f , The relative binding of BMAL1 mutants compared with WT BMAL1, with WT BMAL1 binding to HIF2A set to 1. n = 3 independent experiments. g , HEK293 cells overexpressing WT or mutated Flag-tagged BMAL1 were exposed to ambient hypoxia (1% O 2 ) for 4 h, followed by IP with Flag and blotted with the indicated antibodies. n = 3 independent experiments. h , Quantification of the relative binding in g . n = 3 independent experiments. i , HEK293 cells were transfected with either WT or mutant BMAL1 along with a HIF2A vector. A luciferase reporter assay was performed to evaluate transcriptional activation by the BMAL1–HIF2A complex of the AREG promoter, which contains a shared binding site. n = 4 independent experiments. For f , h and i , data are mean ± s.e.m. j , Schematic illustrating the substantial structural rearrangement of BMAL1 (red) when accommodating various partners to be involved in different pathways. The PAS domains of BMAL1 (red) bend towards nearly opposite direction and position separately when intertwining with HIF2A (purple). Statistical analysis was performed using one-way ANOVA ( f , h and i ). The diagram in j was created using BioRender.

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: Cryo-EM Sample Prep, Binding Assay, Transfection, Mutagenesis, Plasmid Preparation, Luciferase, Reporter Assay, Activation Assay

Journal: Nature

Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury

doi: 10.1038/s41586-025-08898-z

Figure Lengend Snippet: a , Four domain interfaces (I to IV) between BMAL1 and HIF2A. Each of these is indicated by a dashed ellipse. b , Zoom-in views of the interfaces (I to IV) between HIF2A and BMAL1. c , Comparison of the DNA-binding by two bHLH domains in HIF1B/HIF2A (left, PDB ID 4ZPK), BMAL1/HIF2A (middle), and BMAL1/CLOCK (right, PDB ID 4H10). The DNA contacted by the bHLH domains is highlighted in yellow. The PAS domains are omitted for clarity. d , Structural comparison of BMAL1/HIF2A with BMAL1/CLOCK (PDB ID: 4F3L) and HIF1B/HIF2A (PDB ID: 4ZPK). Upper: Structural comparison of BMAL1/HIF2A with BMAL1/CLOCK and HIF1B/HIF2A by aligning bHLH domains (highlighted in blue dashed frames). For clarity, DNA was omitted. Middle: Structure of BMAL1/CLOCK with its BMAL1 PAS-A domain (dashed circle) aligned to that of BMAL1/HIF2A. Lower: Structural comparison of the PAS-B dimers of BMAL1/HIF2A, BMAL1/CLOCK and HIF1B/HIF2A. The conserved residues in the HI loop and their interacting residues are shown. e , Structural comparison of BMAL1/HIF2A and BMAL1/CLOCK by aligning the PAS-A domains of BMAL1. BMAL1 is coloured in red in the BMAL1/HIF2A complex and in grey in the BMAL1/CLOCK complex. f , BMAL1 undergoes structural rearrangements upon binding with various partners. Superimposing the BMAL1/HIF2A and BMAL1/CLOCK complexes by aligning their bHLH domains reveals that BMAL1 (red) undergoes a substantial conformational change, with the two PAS domains bending in nearly opposite directions. BMAL1 exhibits a compact overall architecture when bound with CLOCK (green) and a distinctly separated conformation when interacting with HIF2A (purple).

Article Snippet: Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.

Techniques: Comparison, Binding Assay