BMPR2 Antibody

What is BMPR2 and why is it important in research?

BMPR2 (Bone Morphogenetic Protein Receptor Type 2) is a transmembrane serine/threonine kinase receptor that mediates BMP signaling pathway activities. It forms receptor complexes consisting of two type II and two type I receptors that, upon ligand binding, phosphorylate and activate SMAD transcriptional regulators . BMPR2 is essential for:

• Tissue formation and homeostasis through balanced TGFβ/BMP signaling

• Endothelial cell protection against increased TGFβ responses

• Protection against integrin-mediated mechano-transduction

• Regulation of cellular processes including differentiation, proliferation, and apoptosis

BMPR2 mutations are implicated in pulmonary arterial hypertension (PAH), making it a critical research target for understanding vascular pathologies .

Which applications are most suitable for BMPR2 antibody detection?

BMPR2 antibodies have demonstrated efficacy in multiple applications:

Researchers should determine optimal dilutions for each specific application and validate antibody performance in their experimental system .

How should I select between polyclonal and monoclonal BMPR2 antibodies?

The choice depends on your research objectives:

Polyclonal BMPR2 antibodies:

• Recognize multiple epitopes within BMPR2 (e.g., Rabbit polyclonals targeting aa 250-600 or aa 650-950)

• Offer higher sensitivity for applications like WB and IHC

• Ideal for detecting low-abundance BMPR2 expression

• Better tolerance to protein denaturation

Monoclonal BMPR2 antibodies:

• Target specific epitopes with high specificity (e.g., clone 1F12 or E-1)

• Provide consistent lot-to-lot reproducibility

• Preferable for quantitative analyses and clinical applications

• Reduced background in applications like flow cytometry

For critical comparisons between experimental conditions, monoclonal antibodies offer better consistency, while polyclonal antibodies may provide higher detection sensitivity .

How can I optimize BMPR2 antibody staining in formalin-fixed paraffin-embedded tissues?

Optimizing BMPR2 immunostaining in FFPE tissues requires attention to several parameters:

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is often effective

• Antibody concentration: For goat anti-human BMPR2 antibodies, 15 μg/mL has been successfully used with overnight incubation at 4°C

• Detection system: The Anti-Goat HRP-DAB Cell & Tissue Staining Kit has demonstrated specific labeling of BMPR2 in epithelial cell plasma membranes

• Counterstaining: Hematoxylin provides effective nuclear counterstaining for contrast

• Controls: Include both positive controls (human prostate tissue shows reliable BMPR2 expression) and negative controls (secondary antibody only)

Always validate staining patterns by comparing with established expression patterns in positive control tissues and published literature .

What troubleshooting approaches should I use for BMPR2 western blotting?

When troubleshooting BMPR2 western blotting:

• Protein size verification: BMPR2 should appear at approximately 115-150 kDa depending on post-translational modifications

• Loading control selection: β-actin is commonly used, but consider endothelial-specific controls for endothelial samples

• Signal optimization:

  • Use freshly prepared lysates as BMPR2 can degrade

  • Include protease inhibitors in lysis buffers

  • For weak signals, increase protein loading (50-100 μg) or use enhanced chemiluminescence substrates

• Specificity verification: Confirm specificity using:

  • BMPR2 knockout/knockdown samples as negative controls

  • Multiple antibodies recognizing different epitopes

• Membrane transfer: Ensure complete transfer of high molecular weight BMPR2 by using longer transfer times or specialized transfer conditions for larger proteins

For BMPR2 variants or mutations, adjust gel resolution parameters to distinguish size differences resulting from truncations or post-translational modifications .

How can BMPR2 antibodies help distinguish between different BMPR2 mutation types in PAH research?

BMPR2 mutations in pulmonary arterial hypertension (PAH) can be categorized as:

• NMD+ mutations: Nonsense or frameshift mutations leading to nonsense-mediated decay (NMD) of RNA transcripts, resulting in haploinsufficiency without mutant protein expression

• NMD- mutations: Mutations that bypass NMD, resulting in expression of misfolded proteins that mislocalize intracellularly

Distinguishing these mutation types requires strategic antibody selection:

• Domain-specific antibodies: Use antibodies targeting different domains (N-terminal vs C-terminal) to detect truncated proteins

• Subcellular localization studies: Employ immunofluorescence with BMPR2 antibodies to identify mislocalized mutant BMPR2 proteins in NMD- mutations

• Quantitative analysis: Compare BMPR2 protein levels using western blot densitometry to assess the degree of haploinsufficiency

Research indicates that NMD- mutations may lead to more severe clinical outcomes, with patients developing PAH at an earlier age compared to those with NMD+ mutations . Combined immunoblotting and immunofluorescence approaches with domain-specific antibodies can help characterize these different mutation types in patient-derived samples .

What methodologies can be used to investigate BMPR2-dependent signaling alterations in endothelial cells?

Investigating BMPR2-dependent signaling in endothelial cells requires multi-faceted approaches:

• SMAD pathway activation analysis:

  • Use phospho-specific antibodies against pSMAD1/5 (BMP pathway) and pSMAD2/3 (TGFβ pathway)

  • Compare signaling responses in BMPR2-deficient vs. wild-type cells using western blotting

  • Quantify nuclear translocation of SMADs using immunofluorescence

• Mixed SMAD complex detection:

  • Employ co-immunoprecipitation with BMPR2 antibodies followed by probing for SMAD interaction partners

  • Use proximity ligation assays to detect SMAD complex formation in situ

• Receptor complex analysis:

  • Investigate heteromeric receptor formation (BMPR1/TGFβR1/TGFβR2) in BMPR2-deficient cells

  • Apply small interfering RNA (siRNA) knockdown of TβR2 combined with SMPR2 antibody detection

  • Use selective small-molecule kinase inhibitors (SMKIs) to dissect pathway contributions

• Transcriptional responses:

  • Monitor BMP and TGFβ target genes (ID-3, CTGF) using RT-qPCR

  • Perform RNA-seq analysis to identify global transcriptional changes in BMPR2-deficient cells

These approaches have revealed that BMPR2 deficiency promotes formation of mixed-heteromeric receptor complexes and increased TGFβ responses, potentially driving endothelial-to-mesenchymal transition (EndMT) .

How can BMPR2 antibodies be used to investigate the link between BMPR2 deficiency and inflammation in pulmonary hypertension?

BMPR2 deficiency has been linked to heightened inflammatory responses in pulmonary hypertension through several mechanisms:

• Inflammatory cytokine profiling:

  • Use cell culture supernatants from BMPR2-deficient cells to measure IL-6 and IL-8/KC levels by ELISA

  • Compare cytokine production in BMPR2-deficient vs. wild-type cells after inflammatory stimuli (e.g., LPS)

• Reactive oxygen species (ROS) assessment:

  • Measure superoxide levels in BMPR2-deficient cells using dihydroethidium staining

  • Quantify ROS production in plate-based assays with/without antioxidant treatments

  • Correlate ROS levels with inflammatory cytokine production

• Signaling pathway analysis:

  • Investigate phospho-STAT3 levels in BMPR2-deficient cells using western blotting

  • Examine the loss of extracellular superoxide dismutase (SOD) expression

  • Test the effects of SOD mimetics (e.g., tempol) on inflammatory responses

• In vivo inflammation models:

  • Use BMPR2 antibodies to confirm BMPR2 deficiency in mouse models

  • Analyze lung and circulating cytokine levels after inflammatory challenges

  • Investigate the effects of antioxidant treatment on development of pulmonary hypertension

Research has demonstrated that BMPR2-deficient cells produce higher levels of IL-6 and KC/IL-8 after inflammatory stimulation, and this is associated with increased ROS production. Antioxidant treatments can ameliorate this exaggerated inflammatory response and prevent development of PAH in BMPR2-deficient mice .

What approaches can be used to investigate BMPR2's role in endothelial cell apoptosis and survival?

BMPR2 signaling promotes survival in pulmonary artery endothelial cells, and its loss can lead to increased apoptosis:

• Apoptosis assessment methodologies:

  • Flow cytometry with Annexin V staining to quantify early apoptotic cells

  • TUNEL assay to detect DNA fragmentation in apoptotic cells

  • Measurement of caspase-3 activity as a marker of apoptosis execution

• Experimental design approaches:

  • Compare apoptosis in regular vs. serum-free medium to assess baseline vs. stress-induced apoptosis

  • Add BMP ligands (BMP-2, BMP-7) at 200 ng/mL to evaluate protective effects

  • Include inflammatory stimuli (e.g., TNFα) to model disease conditions

• BMPR2 manipulation strategies:

  • Use siRNA (5 μg) targeting BMPR2 to achieve specific gene silencing

  • Validate knockdown efficiency by RT-PCR and western blotting with BMPR2 antibodies

  • Compare wild-type and BMPR2-deficient endothelial progenitor cells (EPCs)

Research has shown that BMPR2 gene silencing increases apoptosis nearly 3-fold even in the presence of serum. BMP-2 reduces apoptosis induced by serum withdrawal in EPCs from normal subjects but not in EPCs from IPAH patients, supporting the hypothesis that BMPR2 loss-of-function mutations could lead to increased pulmonary EC apoptosis .

How can I design experiments to investigate the relationship between BMPR2 and extracellular matrix remodeling?

BMPR2 deficiency is associated with extracellular matrix remodeling, which can be investigated through:

• ECM protein expression analysis:

  • Use western blotting to quantify levels of ECM proteins such as fibrillin-1 (FBN1) and fibronectin (FN)

  • Perform qRT-PCR to measure transcript levels of ECM genes in BMPR2-deficient vs. wild-type cells

• ECM visualization approaches:

  • Employ immunofluorescence to detect ectopic FBN1 fibers remodeled with fibronectin

  • Focus on cell junctions in BMPR2-deficient endothelial cells to identify abnormal ECM deposits

  • Use confocal microscopy to analyze co-localization patterns

• Integrin activation assessment:

  • Investigate active β1-integrin abundance in integrin-linked kinase (ILK) mechano-complexes

  • Examine co-localization of active integrins with ectopic ECM deposits

  • Use specialized antibodies that recognize activated integrin conformations

• Functional assays:

  • Measure integrin-dependent adhesion and spreading in BMPR2-deficient cells

  • Assess actomyosin-dependent contractility using traction force microscopy

  • Investigate TGFβ retrieval from latent fibrillin-bound depots

These approaches have revealed that BMPR2 deficiency leads to accumulation of ectopic FBN1 fibers in endothelial cell junctions, accompanied by active β1-integrin in mechano-complexes, facilitating retrieval of active TGFβ from its latent deposits and promoting endothelial-to-mesenchymal transition .

What techniques can be employed to investigate BMPR2 receptor complex formation and dynamics?

Investigating BMPR2 receptor complex formation requires sophisticated techniques:

• Co-immunoprecipitation approaches:

  • Use BMPR2 antibodies to pull down receptor complexes

  • Probe for interacting partners (BMP type I receptors, TGFβ receptors) by western blotting

  • Compare receptor associations in different cell types or disease states

• Live-cell imaging techniques:

  • Label BMPR2 with fluorescent tags for real-time visualization

  • Use fluorescence resonance energy transfer (FRET) to detect receptor proximity

  • Employ total internal reflection fluorescence (TIRF) microscopy to focus on membrane dynamics

• Proximity-based detection methods:

  • Implement proximity ligation assays (PLA) to visualize receptor interactions in situ

  • Use bioluminescence resonance energy transfer (BRET) for real-time interaction monitoring

  • Apply cross-linking approaches prior to immunoprecipitation to capture transient complexes

• Functional signaling assessments:

  • Utilize selective small molecule kinase inhibitors (e.g., K02288 against ALK2/ALK1, SB-431542 against ALK5)

  • Combine with siRNA targeting specific receptors (e.g., TβR2)

  • Monitor downstream signaling via phospho-SMAD western blotting

These techniques have revealed that BMPR2 deficiency favors formation of mixed-heteromeric receptor complexes comprising BMPR1, TGFβR1, and TGFβR2, enabling enhanced cellular responses to TGFβ and contributing to disease pathogenesis .

How can BMPR2 antibodies be used in patient-derived samples to study PAH pathogenesis?

BMPR2 antibodies provide valuable tools for investigating PAH pathogenesis in patient samples:

• Tissue immunohistochemistry analysis:

  • Examine BMPR2 expression in pulmonary artery lesions from patients with heritable PAH (HPAH)

  • Look for ectopic fibrillin-1 deposits in proximity to contractile intimal cells

  • Compare BMPR2 expression patterns between mutation carriers and non-carriers

• Patient-derived cell studies:

  • Isolate and culture endothelial cells or smooth muscle cells from PAH patients

  • Create CRISPR/Cas9-modified human EC lines carrying monoallelic BMPR2 mutations

  • Compare signaling responses in patient-derived vs. control cells using BMPR2 antibodies

• Circulating biomarker assessment:

  • Analyze endothelial progenitor cells (EPCs) isolated from normal subjects vs. PAH patients

  • Compare apoptotic responses in the presence and absence of BMPs

  • Use flow cytometry with BMPR2 antibodies to characterize cellular phenotypes

• Genetic-molecular correlations:

  • Correlate BMPR2 protein expression with mutation type (NMD+ vs. NMD-)

  • Examine post-translational modifications of BMPR2 using phospho-specific antibodies

  • Investigate compensatory mechanisms in BMPR2-deficient cells

These approaches have revealed that BMPR2-deficient heritable PAH patients show ectopic fibrillin-1 deposits in pulmonary artery lesions, and their cells exhibit increased TGFβ signaling and enhanced inflammatory responses .

What methodologies can help identify potential therapeutic targets in BMPR2-deficient models?

Identifying therapeutic targets in BMPR2-deficient models requires systematic approaches:

• Pathway analysis for target identification:

  • Use RNA-Seq to identify differentially regulated genes in BMPR2-deficient cells

  • Focus on altered TGFβ/BMP signaling components, inflammatory mediators, and ECM proteins

  • Validate key targets using BMPR2 antibodies and pathway-specific tools

• Pharmacological intervention testing:

  • Evaluate antioxidant treatments (e.g., tempol) to counter increased ROS production

  • Test selective small molecule kinase inhibitors targeting ALK5 to inhibit excessive TGFβ signaling

  • Assess anti-inflammatory compounds to reduce cytokine production

• Integrin-targeting strategies:

  • Investigate integrin-blocking antibodies to prevent excessive ECM engagement

  • Evaluate inhibitors of integrin-linked kinase (ILK) mechano-complexes

  • Test compounds that reduce actomyosin-dependent contractility

• Combination approaches:

  • Implement dual pathway inhibition (TGFβ pathway + inflammatory mediators)

  • Combine antioxidant treatment with targeted pathway modulation

  • Use BMPR2 antibodies to monitor treatment effects on receptor expression