Phospho-PDGFRB (Y740) Recombinant Monoclonal Antibody

What is PDGFRB and what is the significance of Y740 phosphorylation?

PDGFRB (Platelet-derived growth factor receptor beta) is a tyrosine-protein kinase that functions as a cell-surface receptor for homodimeric PDGFB and PDGFD, as well as heterodimers formed by PDGFA and PDGFB. It plays essential roles in regulating embryonic development, cell proliferation, survival, differentiation, chemotaxis, and migration .

The Y740 phosphorylation site is one of several key tyrosine residues that become phosphorylated upon ligand binding. This specific phosphorylation event contributes to the activation of downstream signaling cascades, including the recruitment and activation of signaling molecules such as PLCG1, PIK3R1, PTPN11, RASA1/GAP, CBL, SHC1, and NCK1 . Phosphorylation at Y740 is particularly important for the initiation of PI3K pathway activation, which leads to AKT signaling and promotes cell survival and proliferation.

The commercially available Phospho-PDGFRB (Y740) antibodies have been validated for use with human samples , while some products also demonstrate reactivity with mouse and rat samples . When working with these antibodies, researchers should confirm species cross-reactivity based on the specific product documentation.

For in vitro studies, HeLa cells have been used for validation in proximity ligation assays , but many other cell types that express PDGFRB can be suitable study models, particularly those of mesenchymal origin such as fibroblasts, vascular smooth muscle cells, and pericytes.

What is the recommended protocol for detecting Phospho-PDGFRB (Y740) using Western blot?

For optimal Western blot detection of Phospho-PDGFRB (Y740):

• Prepare cell lysates immediately after treatment, using phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, and β-glycerophosphate) in the lysis buffer to preserve phosphorylation status.

• Use SDS-PAGE with 7-8% gels (PDGFRB is approximately 180-190 kDa).

• Transfer proteins to PVDF membranes (preferred over nitrocellulose for phospho-proteins).

• Block with 5% BSA in TBST (not milk, which contains phospho-proteins).

• Incubate with primary Phospho-PDGFRB (Y740) antibody at recommended dilution (typically 1:1000) overnight at 4°C.

• Wash thoroughly with TBST (at least 3 times for 5-10 minutes each).

• Incubate with appropriate HRP-conjugated secondary antibody.

• Develop using enhanced chemiluminescence reagents.

• As a loading control, strip and reprobe with total PDGFRB antibody to calculate the phosphorylation ratio .

How can Proximity Ligation Assay enhance detection of Phospho-PDGFRB (Y740)?

The Proximity Ligation Assay (PLA) offers superior sensitivity for detecting phosphorylated proteins, particularly when phosphorylation levels are low or when studying single-cell heterogeneity:

• The assay uses two antibodies: one specific to phosphorylated Y740 site (rabbit polyclonal) and another recognizing total PDGFRB protein (mouse monoclonal) .

• Secondary antibodies conjugated with oligonucleotides (PLA probes) bind to the primary antibodies.

• When the two PLA probes are in close proximity (<40 nm), the oligonucleotides can interact through a subsequent addition of two other circle-forming DNA oligonucleotides.

• After ligation of the oligonucleotides by a DNA ligase, they are amplified via rolling circle amplification using a DNA polymerase.

• Fluorescently labeled oligonucleotides hybridize to the amplification product, resulting in visible fluorescent dots where phosphorylated PDGFRB is present .

This method provides single-molecule resolution and enables visualization of individual phosphorylated PDGFRB proteins, represented as distinct red dots that can be quantified using image analysis software such as BlobFinder from Uppsala University .

What are the advantages of cell-based ELISA for PDGFRB phosphorylation studies?

Cell-based ELISA offers several advantages for studying PDGFRB phosphorylation:

• High-throughput capability: Performed in 96-well microplates, enabling screening of multiple conditions simultaneously.

• Quantitative analysis: Provides more precise quantification than Western blot.

• Time efficiency: Much faster than traditional Western blot analysis.

• Conservation of reagents: Requires smaller amounts of cells and treatment compounds.

• Normalization capability: Crystal violet staining allows normalization to cell number, adjusting for differences in cell density between wells .

The typical workflow involves:

• Culturing cells directly in 96-well plates

• Treating cells with stimulants/inhibitors

• Fixing and permeabilizing cells

• Incubating with anti-Phospho-PDGFRB (Y740) antibody

• Detecting with HRP-conjugated secondary antibody

• Measuring colorimetric signal with standard ELISA reader

• Normalizing to cell number using crystal violet staining

How can researchers differentiate between direct and indirect phosphorylation of PDGFRB at Y740?

Distinguishing between direct PDGFRB autophosphorylation and trans-phosphorylation by other kinases requires sophisticated experimental approaches:

• In vitro kinase assays: Using recombinant PDGFRB with and without its kinase domain, exposed to potential trans-phosphorylating kinases, followed by immunoblotting with Phospho-PDGFRB (Y740) antibodies.

• Kinase-dead mutants: Comparing Y740 phosphorylation in cells expressing wild-type versus kinase-dead (K634R) PDGFRB mutants following stimulation.

• Selective kinase inhibitors: Applying specific inhibitors of PDGFRB kinase activity versus inhibitors of other potential upstream kinases, then measuring Y740 phosphorylation.

• Temporal dynamics analysis: Monitoring the phosphorylation kinetics at Y740 relative to other autophosphorylation sites, as autophosphorylation and trans-phosphorylation often follow different time courses.

• Proximity ligation assays: Detecting physical associations between PDGFRB and potential trans-phosphorylating kinases in conjunction with Y740 phosphorylation.

What is the relationship between PDGFRB Y740 phosphorylation and downstream signaling pathways?

Y740 phosphorylation of PDGFRB initiates specific downstream signaling events:

• Y740 phosphorylation creates binding sites for proteins containing SH2 domains, particularly the p85 regulatory subunit of PI3K (PIK3R1) .

• The binding of p85 leads to activation of PI3K, resulting in the generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane.

• PIP3 recruits and facilitates the activation of AKT/PKB through phosphorylation by PDK1 and mTORC2.

• Activated AKT promotes cell survival, proliferation, and metabolism through numerous downstream targets including:

  • Inhibition of pro-apoptotic factors (BAD, caspase-9)

  • Activation of mTORC1 signaling

  • Inhibition of FOXO transcription factors

  • Regulation of glucose metabolism

Compared to other phosphorylation sites on PDGFRB, Y740 phosphorylation is particularly important for the PI3K-AKT axis, while other phosphorylated tyrosines (e.g., Y751, Y771, Y1009, Y1021) preferentially activate different pathways such as SRC, PLCγ, SHP2, and RAS-MAPK signaling.

How can PDGFRB Y740 phosphorylation be modulated experimentally?

Researchers can manipulate PDGFRB Y740 phosphorylation through several approaches:

When designing such experiments, it is crucial to include appropriate controls and time course analyses, as PDGFRB phosphorylation is often dynamic and context-dependent.

What are common issues when working with Phospho-PDGFRB (Y740) antibodies and how can they be resolved?

For optimal results, store antibodies at -20°C or lower, aliquot to avoid repeated freeze-thaw cycles, and return reagents to -20°C storage immediately after use .

How should researchers validate the specificity of Phospho-PDGFRB (Y740) antibodies?

Comprehensive validation of Phospho-PDGFRB (Y740) antibodies should include:

• Peptide competition assays: Pre-incubating the antibody with phospho-Y740 peptide should eliminate specific signal.

• Phosphatase treatment controls: Treating samples with lambda phosphatase should abolish phospho-specific signal while preserving total PDGFRB detection.

• Stimulation/inhibition experiments: PDGF-BB stimulation should increase signal; pre-treatment with PDGFR inhibitors should decrease signal.

• Y740F mutant expression: Cells expressing PDGFRB with tyrosine-to-phenylalanine mutation at position 740 should not show phospho-specific signal.

• Knockout/knockdown controls: PDGFRB-null or knockdown cells should not display specific signal.

• Multi-method confirmation: Consistent results across different techniques (Western blot, ELISA, immunofluorescence) strengthen confidence in antibody specificity.

How can Phospho-PDGFRB (Y740) detection be integrated into multiplexed phosphoprotein analysis?

Advanced multiplexed approaches for studying PDGFRB phosphorylation in broader signaling contexts include:

• Multiplex bead-based assays: Using differentially coded beads, each coupled with antibodies against different phosphoproteins including Phospho-PDGFRB (Y740).

• Mass cytometry (CyTOF): Leveraging metal-tagged antibodies for simultaneous detection of multiple phosphorylation sites, including PDGFRB Y740, alongside other cellular markers.

• Sequential stripping and reprobing: Systematic analysis of multiple phosphoproteins on the same Western blot membrane by stripping and reprobing.

• Multiplex immunofluorescence: Using primary antibodies from different species and spectrally distinct fluorophores to simultaneously visualize PDGFRB Y740 phosphorylation alongside other phosphoproteins.

• Proximity extension assays: Combining Phospho-PDGFRB (Y740) detection with other protein assays in multiplexed proteomic profiling.

When designing multiplexed experiments, careful antibody selection is crucial to avoid cross-reactivity and ensure compatible assay conditions for all target phosphoproteins.

What are the implications of PDGFRB Y740 phosphorylation in disease models and potential therapeutic applications?

PDGFRB Y740 phosphorylation has significant implications in various disease contexts:

• Cancer: Aberrant PDGFRB signaling, including Y740 phosphorylation, contributes to oncogenesis in multiple tumor types, particularly those of mesenchymal origin. Monitoring Y740 phosphorylation can provide insights into:

  • Tumor cell proliferation and survival

  • Angiogenesis mechanisms

  • Efficacy of tyrosine kinase inhibitors

  • Resistance mechanisms to targeted therapies

• Fibrotic diseases: PDGFRB signaling drives fibroblast activation and extracellular matrix production in:

  • Pulmonary fibrosis

  • Liver fibrosis

  • Renal fibrosis

  • Cardiac fibrosis

• Vascular disorders: Y740 phosphorylation mediates vascular remodeling in:

  • Atherosclerosis

  • Pulmonary hypertension

  • Restenosis after vascular injury

• Neurodegenerative conditions: PDGFRB signaling affects blood-brain barrier integrity and neuroinflammation.

Therapeutic strategies targeting PDGFRB phosphorylation include tyrosine kinase inhibitors, neutralizing antibodies against PDGF ligands, and peptide mimetics that interfere with specific phosphotyrosine-dependent protein interactions.

How might single-cell analysis of PDGFRB Y740 phosphorylation advance our understanding of cellular heterogeneity?

Single-cell approaches for analyzing PDGFRB Y740 phosphorylation can provide unprecedented insights into cellular heterogeneity:

• Single-cell phosphoproteomics: Mass spectrometry-based approaches can reveal cell-to-cell variation in PDGFRB phosphorylation states.

• Microfluidic platforms: These enable analysis of Y740 phosphorylation in individual cells under controlled microenvironments.

• Proximity ligation assay with digital quantification: The distinct fluorescent dots produced in proximity ligation assays can be counted in individual cells to provide absolute quantification of phosphorylated PDGFRB molecules .

• Live-cell biosensors: FRET-based reporters for monitoring PDGFRB phosphorylation dynamics in living cells in real-time.

• Spatial transcriptomics integration: Combining Phospho-PDGFRB (Y740) detection with spatial transcriptomics can reveal relationships between PDGFRB phosphorylation and transcriptional programs in tissue contexts.

These approaches will help elucidate how heterogeneity in PDGFRB phosphorylation contributes to diverse cellular behaviors within seemingly homogeneous populations, with implications for understanding disease progression and treatment response variability.

What computational approaches can enhance the analysis of PDGFRB phosphorylation data?

Advanced computational methods are increasingly important for analyzing complex PDGFRB phosphorylation data:

• Machine learning algorithms: Can identify patterns in large datasets associating PDGFRB Y740 phosphorylation with cellular outcomes.

• Network analysis: Places PDGFRB Y740 phosphorylation within broader signaling networks to predict system-wide effects.

• Kinetic modeling: Mathematical models describing the dynamics of PDGFRB phosphorylation and downstream signaling can generate testable predictions about pathway regulation.

• Image analysis software: Tools like BlobFinder enable quantitative analysis of proximity ligation assay results, counting individual phosphorylated PDGFRB molecules .

• Integrative multi-omics approaches: Combining phosphoproteomic data with transcriptomic, metabolomic, and other datasets to build comprehensive models of PDGFRB-mediated cellular responses.

These computational approaches not only enhance data analysis but also guide experimental design by identifying key parameters and timepoints for optimal investigation of PDGFRB phosphorylation biology.