Phospho-CSF1R (Y561) Antibody

What is CSF1R and what is the significance of the Y561 phosphorylation site?

CSF1R (Colony Stimulating Factor 1 Receptor) is a tyrosine kinase transmembrane receptor belonging to the CSF1/PDGF receptor family. This receptor mediates most, if not all, biological effects of colony stimulating factor 1, a cytokine that controls the production, differentiation, and function of macrophages . The Y561 residue is a critical phosphorylation site that binds to SRC and other Src Family Kinases (SFKs), playing a crucial role in disrupting cell-cell adhesion . This phosphorylation site serves as an important regulatory point in CSF1R-mediated signaling cascades.

Ligand binding activates the receptor kinase through oligomerization and transphosphorylation processes. Mutations in CSF1R have been associated with predisposition to myeloid malignancy . Understanding Y561 phosphorylation is essential for elucidating the mechanisms of CSF1R function in both normal cellular processes and disease states.

What are the recommended applications for Phospho-CSF1R (Y561) antibodies?

Phospho-CSF1R (Y561) antibodies are versatile tools applicable in multiple experimental techniques. Based on manufacturer specifications, these antibodies are suitable for:

• Western Blot (WB) analysis (1:500-1:1000 dilution)

• Immunohistochemistry on paraffin-embedded sections (IHC-P) (1:50-1:100 dilution)

• Immunohistochemistry with PFA-fixed samples

These applications enable researchers to detect endogenous levels of CSF1R specifically when phosphorylated at tyrosine 561, making them valuable for studying activation states of the receptor in various cellular contexts .

What controls should be included when working with Phospho-CSF1R (Y561) antibodies?

When conducting experiments with Phospho-CSF1R (Y561) antibodies, several controls should be implemented:

• Positive control: Cell extracts treated with PMA (phorbol 12-myristate 13-acetate) have been validated for detecting phosphorylated CSF1R at Y561 . HepG2 cells treated with PMA serve as an excellent positive control for Western blot applications.

• Negative control: Samples treated with phosphatase or cells expressing a Y561F mutant form of CSF1R can serve as negative controls, as they should show significantly reduced or absent signal .

• Loading control: Include antibodies against total CSF1R to normalize phosphorylation levels and ensure equal protein loading.

• Peptide competition assay: Pre-incubation of the antibody with the immunizing phosphopeptide can confirm specificity by blocking antibody binding to the target.

These controls help validate antibody specificity and ensure experimental rigor when studying CSF1R phosphorylation states.

How does the Y561F mutation affect CSF1R signaling and function?

• SRC-dependent sites: These show increased phosphorylation in cells expressing wildtype CSF1R compared to those with Y561F mutation, indicating their dependence on SRC recruitment to Y561.

• SRC-independent sites: These sites display no significant differences in phosphorylation between wildtype and Y561F CSF1R, suggesting alternative regulatory mechanisms.

• SRC-inhibited sites: These exhibit higher phosphorylation in Y561F mutant cells, indicating that SRC recruitment normally suppresses their phosphorylation.

Interestingly, the Y561F mutation does not affect auto-phosphorylation of constitutively active CSF1R (CA-CSF1R) at key sites including Y809 (activation loop), Y873, Y699 (Grb2 binding site), and Y923 . This suggests that the total kinase activity remains intact despite the mutation.

What are the known SRC-dependent substrates downstream of CSF1R Y561 phosphorylation?

Quantitative mass spectrometry analyses have identified several SRC-dependent substrates downstream of CSF1R Y561 phosphorylation. These proteins show increased phosphorylation in cells expressing wildtype CSF1R compared to those expressing the Y561F mutant:

Additional SRC-dependent phosphorylation sites identified include p120, ACK, Hrs, β-adaptin, intersectin 2, and KIAA1217 . Several other proteins have supporting evidence for SRC-dependent regulation, including Integrin β4 Y1510, FLJ20625 Y40, Hrs Y334, TJAP1 Y352, and ZDHHC7 Y130 .

These findings highlight the diverse range of cellular processes influenced by CSF1R-mediated SRC activation, including transcriptional regulation, receptor trafficking, cytoskeletal organization, and cell adhesion.

How can mass spectrometry be used to identify novel substrates dependent on CSF1R Y561 phosphorylation?

Quantitative mass spectrometry offers a powerful approach for identifying novel CSF1R Y561-dependent phosphorylation events. A comprehensive methodology based on the search results includes:

• SILAC labeling: Metabolically label cells expressing wildtype CSF1R with "heavy" isotopic amino acids (13C6-15N2-L-lysine and 13C6-15N4-L-arginine) and cells expressing Y561F mutant with "light" isotopic variants . This allows direct quantitative comparison of phosphopeptides.

• Sample preparation: Culture cells under appropriate conditions (e.g., EGF starvation to ensure dominant CSF1R signaling), mix samples in a 1:1 "heavy/light" ratio, and perform proteolytic digestion .

• Phosphotyrosine enrichment: Conduct immunoprecipitation with anti-phosphotyrosine antibodies to enrich tyrosine-phosphorylated peptides prior to LC-MS/MS analysis .

• Quantitative analysis: Calculate fold differences in tyrosine phosphorylated peptides by determining the ratio of peptides detected in each cell pair. Establish thresholds for defining upregulation (e.g., >1.55-fold change), unchanged (0.63-1.55), and downregulation (<0.63) .

• Validation: Confirm selected hits using orthogonal methods such as Western blotting with phosphosite-specific antibodies, mutational analysis, or functional assays.

This approach successfully identified 275 phosphopeptides in the comparison of CA-CSF-1R MCF-10A cells to parental MCF-10A cells, and 324 phosphopeptides when comparing CA-CSF-1R to CA-CSF-1R Y561F cells , demonstrating its efficacy for discovering novel phosphorylation events in CSF1R signaling.

What is the relationship between CSF1R Y561 phosphorylation and epithelial cell biology?

CSF1R Y561 phosphorylation plays significant roles in epithelial cell biology, particularly in regulating cell-cell adhesion and epithelial-to-mesenchymal transition (EMT) processes:

• Disruption of cell-cell adhesion: The Y561 phosphorylation site has been identified as critical for disrupting cell-cell adhesion in epithelial cells . This function is likely mediated through SRC recruitment and subsequent phosphorylation of adhesion components.

• Regulation of epithelial markers: Studies in MCF-10A mammary epithelial cells expressing constitutively active CSF1R show alterations in epithelial markers dependent on Y561 phosphorylation, suggesting a role in maintaining epithelial integrity.

• Effects on junction proteins: Known SRC-dependent substrates downstream of CSF1R include p120, which is a critical component of adherens junctions. Phosphorylation of p120 can alter its binding to cadherins and influence junction stability .

• Influence on cell morphology: CSF1R activation in epithelial cells can induce morphological changes resembling EMT, with the Y561 site playing a key role in this transition through recruitment of SRC family kinases.

Understanding these relationships is particularly important in the context of epithelial cancers, where aberrant CSF1R signaling may contribute to increased invasiveness and metastatic potential through disruption of normal epithelial architecture.

How can researchers effectively validate the specificity of Phospho-CSF1R (Y561) antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For Phospho-CSF1R (Y561) antibodies, a comprehensive validation approach should include:

• Peptide competition assay: Pre-incubate the antibody with the synthetic phosphopeptide used as immunogen (corresponding to residues surrounding Y561 of human CSF1R) . Signal elimination or reduction confirms specificity for the phospho-epitope.

• Phosphatase treatment: Treat half of your sample with lambda phosphatase prior to immunoblotting. Disappearance of signal in the treated sample confirms phospho-specificity.

• Genetic validation: Compare signal between wildtype cells and those expressing Y561F mutant CSF1R or CRISPR/Cas9-mediated CSF1R knockout cells. Absence of signal in mutant or knockout cells supports antibody specificity.

• Stimulation experiments: Compare unstimulated cells with those treated with known CSF1R activators (like CSF1 or PMA) . Increased signal after stimulation provides functional validation.

• Cross-reactivity assessment: Test the antibody on samples from multiple species if working with non-human models, considering that the available antibodies recognize both human and mouse CSF1R phosphorylated at Y561 .

• Multiple detection methods: Validate findings using at least two different techniques (e.g., Western blot and immunohistochemistry) to ensure consistent results across platforms.

What are the optimal working dilutions and conditions for different applications of Phospho-CSF1R (Y561) antibodies?

Optimal working dilutions vary by application and should be determined empirically for each experimental system. Based on manufacturer recommendations:

Additional considerations for optimal results include:

• Sample preparation: For Western blotting, include phosphatase inhibitors in lysis buffers to preserve phosphorylation status.

• Blocking conditions: Use BSA rather than milk for blocking membranes when detecting phosphoproteins, as milk contains phosphoproteins that may increase background.

• Antibody diluent: Prepare antibodies in buffer containing BSA and phosphatase inhibitors to maintain stability and specificity.

• Incubation times: For Western blots, overnight incubation at 4°C often yields better results than shorter incubations at room temperature.

The optimal working dilution should ultimately be determined by the end-user based on their specific experimental conditions and sample types .

How should Phospho-CSF1R (Y561) antibodies be stored to maintain activity?

Proper storage is essential for maintaining antibody activity and specificity. For Phospho-CSF1R (Y561) antibodies:

• Storage temperature: Store at -20°C for long-term preservation of activity . Avoid repeated freeze-thaw cycles by preparing working aliquots upon first thaw.

• Formulation: The antibodies are typically supplied in PBS (without Mg2+ and Ca2+), 150mM NaCl, pH 7.4, with 50% glycerol and 0.02% sodium azide . This formulation helps maintain stability during storage.

• Aliquoting: Divide the stock solution into small aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality and reduce specificity .

• Working solution handling: Once diluted for use, keep antibody solutions on ice and use within 24 hours for optimal performance.

• Shipping and temporary storage: While antibodies can tolerate brief periods at higher temperatures during shipping, they should be transferred to -20°C immediately upon receipt.

• Expiration tracking: Document the date of first use and monitor performance over time, as antibody activity may diminish even with proper storage.

Following these storage recommendations will help ensure consistent experimental results and maximize the usable lifetime of the antibody.

What strategies can improve detection sensitivity when working with Phospho-CSF1R (Y561) antibodies?

Several strategies can enhance detection sensitivity when working with Phospho-CSF1R (Y561) antibodies:

• Enrichment of phosphoproteins: Prior to Western blotting, consider using phosphotyrosine immunoprecipitation to concentrate phosphorylated proteins from your sample, similar to the approach used in mass spectrometry studies .

• Signal amplification systems: Utilize enhanced chemiluminescence (ECL) substrates with higher sensitivity or consider fluorescent secondary antibodies with direct digital imaging for improved signal-to-noise ratio.

• Sample preparation optimization:

  • Include fresh phosphatase inhibitors in all buffers

  • Use rapid lysis methods to minimize dephosphorylation during processing

  • Consider stimulating cells with appropriate activators (such as PMA) to increase phosphorylation levels

• Optimization of transfer conditions: For Western blots, adjust transfer conditions (time, voltage, buffer composition) based on the molecular weight of CSF1R (~170 kDa) to ensure efficient transfer to membranes.

• Detection system selection: For IHC applications, compare different detection systems (ABC, polymer-based) to determine which provides optimal signal with minimal background.

• Antigen retrieval optimization: For IHC applications, test different antigen retrieval methods to maximize epitope accessibility while preserving tissue morphology.

• Secondary antibody selection: Choose high-affinity, application-specific secondary antibodies that match your detection system for maximal sensitivity.

Implementing these strategies can significantly improve detection sensitivity and enable visualization of even low levels of CSF1R Y561 phosphorylation in experimental samples.

How can Phospho-CSF1R (Y561) antibodies be used to study SRC-dependent and SRC-independent signaling pathways?

Phospho-CSF1R (Y561) antibodies can be powerful tools for dissecting SRC-dependent and SRC-independent signaling pathways through several experimental approaches:

• Comparative phosphoproteomic analysis: Use the antibody to immunoprecipitate phosphorylated CSF1R complexes from cells expressing wildtype versus Y561F mutant receptors, followed by mass spectrometry to identify differentially associated proteins and phosphorylation sites .

• Temporal signaling dynamics: Conduct time-course experiments following CSF1R activation, using the antibody to monitor Y561 phosphorylation in parallel with downstream substrates identified as SRC-dependent (e.g., STAT5A, EphB4) and SRC-independent (e.g., ERK1/2, p38-alpha) .

• Inhibitor studies: Compare the effects of CSF1R-specific inhibitors versus SRC family kinase inhibitors on Y561 phosphorylation and downstream signaling events to distinguish direct CSF1R-mediated effects from those requiring SRC activity.

• Co-immunoprecipitation: Use the antibody in conjunction with SRC-specific antibodies to isolate and characterize CSF1R-SRC complexes and identify additional components of the signaling complex.

• Functional validation: Combine antibody-based detection with functional assays (migration, proliferation, differentiation) in the presence of pathway-specific inhibitors to correlate signaling events with biological outcomes.

This multi-faceted approach can help researchers delineate the complex network of SRC-dependent and SRC-independent signaling events downstream of CSF1R activation, as suggested by the identification of distinct sets of phosphorylation events in previous studies .

What experimental approaches can be used to study the role of CSF1R Y561 phosphorylation in cell-cell adhesion?

To investigate the role of CSF1R Y561 phosphorylation in cell-cell adhesion, researchers can employ several complementary approaches:

• Comparison of wildtype versus Y561F mutant CSF1R: Express wildtype or Y561F mutant CSF1R in epithelial cell models and assess:

  • Localization and stability of adherens junction proteins (E-cadherin, β-catenin, p120) by immunofluorescence and biochemical fractionation

  • Transepithelial electrical resistance (TEER) measurements to quantify barrier function

  • Calcium-switch assays to monitor junction assembly and disassembly dynamics

• Live-cell imaging: Perform time-lapse microscopy of fluorescently tagged junction proteins in cells expressing wildtype versus Y561F CSF1R to track real-time changes in junction dynamics following receptor activation.

• Rescue experiments: In cells expressing Y561F mutant CSF1R, test whether constitutively active SRC can rescue adhesion defects, confirming the SRC-dependence of observed phenotypes.

• Proximity ligation assays (PLA): Use PLA to detect and quantify interactions between phosphorylated CSF1R and junction components or SRC in situ, providing spatial information about signaling events.

• Domain-specific mutations: Introduce mutations in downstream substrates at SRC phosphorylation sites (such as p120) to determine which specific phosphorylation events mediate CSF1R effects on adhesion.

• 3D culture models: Assess the impact of Y561 phosphorylation on epithelial morphogenesis in 3D culture systems, which better recapitulate tissue architecture than monolayer cultures.

These approaches can help elucidate the mechanisms by which CSF1R Y561-mediated SRC recruitment and activation influence cell-cell adhesion, a critical process in both normal tissue homeostasis and pathological conditions like cancer metastasis .

How can researchers distinguish between CSF1R Y561 phosphorylation and other tyrosine phosphorylation events in complex samples?

Distinguishing specific phosphorylation events in complex samples requires careful experimental design and appropriate controls:

• Use of phosphosite-specific antibodies: Employ antibodies that specifically recognize CSF1R phosphorylated at Y561, like those described in the search results , which have been validated to detect endogenous levels of CSF1R only when phosphorylated at this specific tyrosine residue.

• Mutant controls: Include samples from cells expressing Y561F mutant CSF1R as negative controls to confirm antibody specificity in your experimental system .

• Sequential immunoprecipitation: First immunoprecipitate total CSF1R, then probe with anti-phosphotyrosine antibodies, or vice versa, to confirm the identity of phosphorylated proteins.

• Molecular weight verification: CSF1R has a distinctive molecular weight (~170 kDa), which helps distinguish it from other phosphorylated proteins on Western blots.

• Phosphopeptide mapping: For mass spectrometry applications, ensure rigorous identification criteria including:

  • High mass accuracy measurements

  • Detection of diagnostic fragment ions in MS/MS spectra

  • Comparison of retention times with synthetic phosphopeptide standards

• Multiple antibody validation: Use complementary approaches such as combining a general anti-phosphotyrosine antibody with CSF1R-specific antibodies to confirm findings.

• Receptor activation controls: Include samples with and without CSF1R activation (by ligand stimulation or use of constitutively active mutants) to distinguish activation-dependent phosphorylation events.

These approaches collectively provide confidence in the specific detection of CSF1R Y561 phosphorylation, even in complex biological samples.

What are common challenges when working with Phospho-CSF1R (Y561) antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with phospho-specific antibodies like Phospho-CSF1R (Y561):

• Low signal intensity:

  • Challenge: Phosphorylation events are often transient and present at low stoichiometry.

  • Solution: Enrich phosphoproteins through immunoprecipitation before Western blotting; use signal amplification systems; optimize stimulation conditions to maximize phosphorylation .

• High background:

  • Challenge: Non-specific binding can obscure specific signals.

  • Solution: Optimize blocking conditions (use BSA instead of milk); increase washing stringency; titrate primary antibody concentration; use phosphopeptide competition controls to distinguish specific from non-specific signals .

• Sample degradation:

  • Challenge: Phosphorylation status can change rapidly during sample processing.

  • Solution: Include phosphatase inhibitors in all buffers; process samples quickly; keep samples cold throughout preparation; consider phosphatase treatment of control samples as a validation approach.

• Cross-reactivity:

  • Challenge: Antibodies may recognize similar phosphoepitopes on other proteins.

  • Solution: Include Y561F mutant controls ; perform peptide competition assays; validate results using orthogonal methods.

• Reproducibility issues:

  • Challenge: Results may vary between experiments or antibody lots.

  • Solution: Standardize protocols; include consistent positive controls (e.g., PMA-treated HepG2 cells) ; consider purchasing larger antibody lots for long-term studies.

• Species differences:

  • Challenge: Antibody performance may vary between human and mouse samples despite recognized homology.

  • Solution: Validate antibody performance in your specific model system; consider species-specific positive controls.

Addressing these challenges through careful experimental design and appropriate controls will improve the reliability and reproducibility of results obtained with Phospho-CSF1R (Y561) antibodies.