Phospho-SMAD5 (S463+S465) Recombinant Monoclonal Antibody

What is the biological significance of SMAD5 phosphorylation at S463/S465?

Phosphorylation of SMAD5 at serine residues 463 and 465 represents a critical activation event in BMP (Bone Morphogenetic Protein) signaling. When BMP ligands bind to type I and type II serine/threonine kinase receptors, the type I receptors (ALK1, 2, 3, and 6) phosphorylate SMAD5 at these specific residues. This phosphorylation enables SMAD5 to form complexes with SMAD4 and translocate to the nucleus, where it regulates gene expression involved in development, cell proliferation, and differentiation . Particularly in hematopoietic cells, SMAD5 activation by BMP4 significantly increases the proliferation of erythroid progenitors and formation of glycophorin-A+ cells . This phosphorylation event serves as a fundamental mechanism for regulating both BMP and TGF-β signaling pathways, controlling gene expression, and coordinating cellular processes during development and tissue homeostasis .

How does phosphorylated SMAD5 differ from non-phosphorylated SMAD5 in cellular function?

The phosphorylation state of SMAD5 dramatically alters its cellular function and localization:

• Phosphorylated SMAD5 (pSMAD5): Forms complexes with SMAD4 and predominantly functions in the nucleus as a transcription regulator, activating target genes involved in development and differentiation .

• Non-phosphorylated SMAD5: Recent research indicates significant cytoplasmic roles independent of its transcriptional activity. Non-phosphorylated SMAD5 interacts with hexokinase 1 (HK1), a rate-limiting enzyme in glycolysis, enhancing its activity and cellular energy production . It also responds to changes in intracellular pH (pHi), functioning as a pH messenger that maintains bioenergetic homeostasis .

This dual functionality makes SMAD5 unique among SMAD proteins, as it exerts biological effects through both canonical (phosphorylation-dependent, nuclear) and non-canonical (cytoplasmic) mechanisms .

What types of stimuli induce SMAD5 phosphorylation at S463/S465?

Several stimuli can induce SMAD5 phosphorylation at S463/S465:

• BMP ligands: BMP2 and BMP4 are potent inducers of SMAD5 phosphorylation, with dose-dependent effects. Studies show concentration-dependent phosphorylation occurring within 30 minutes of exposure .

• TGF-β: While primarily associated with SMAD2/3 activation, TGF-β also induces SMAD1/5 phosphorylation in various epithelial cells, including normal mammary epithelial lines (NMuMG, EpH4, MCF-10A), transformed mammary cell lines (MCF-7, MDA-MB-231), and pancreatic adenocarcinomas .

• Viral infection: Some viral infections, such as Rift Valley fever virus, can trigger SMAD protein phosphorylation, including SMAD1/5 at S463/S465 .

• Inorganic compounds: Certain ions, such as zirconium, can up-regulate the BMP/SMAD signaling pathway, resulting in increased phosphorylation of SMAD1/5 at S463/S465 .

What are the optimal conditions for inducing and detecting SMAD5 phosphorylation in cell culture models?

For optimal induction and detection of SMAD5 phosphorylation:

Induction protocol:

• BMP stimulation: Treat cells with 1-30 ng/mL of recombinant BMP4 or BMP2 for 30 minutes at 37°C .

• Cell density: Seed cells at a density of 0.2-1.0 × 10^6 cells/mL for suspension cells .

• Serum starvation: For cleaner results, serum-starve cells for 4-6 hours before stimulation.

Detection conditions:

• Fixation: Fix cells with 4% paraformaldehyde for 20 minutes .

• Antibody dilution: For Western blotting, use 1:500-1:5000 dilution; for immunohistochemistry, use 1:50-1:200; for immunofluorescence, use 1:100-1:400 .

• Incubation: For maximum sensitivity, incubate with primary antibody overnight at 2-8°C .

Controls to include:

• Unstimulated cells (negative control)

• BMP receptor inhibitor (e.g., DMH-1) pre-treatment (inhibition control)

• Total SMAD5 measurement (normalization control)

The Cell-Based ELISA protocol has been validated to provide reliable results when following these parameters, with fluorogenic detection at 600 nm for phosphorylated SMAD1/5 and 450 nm for a housekeeping protein such as GAPDH for normalization .

How should researchers compare phospho-SMAD5 detection across different experimental techniques?

Each detection method has unique advantages and limitations when measuring phospho-SMAD5:

For cross-method validation:

• Always normalize phospho-SMAD5 to total SMAD5 or a housekeeping protein

• Use the same stimulation conditions across techniques

• Apply matched antibody clones when possible

• Include appropriate negative and positive controls for each method

What are the critical considerations when selecting a phospho-SMAD5 (S463/S465) antibody for specific applications?

When selecting a phospho-SMAD5 antibody, consider these critical factors:

Specificity considerations:

• Cross-reactivity: Many antibodies recognize both phospho-SMAD1 (S463/S465) and phospho-SMAD5 (S463/S465) due to sequence homology. Some also detect phospho-SMAD9 (S465/S467). Check if your research requires SMAD5-specific detection .

• Non-specific binding: Validate using knockout/knockdown controls or blocking peptides.

Application-specific parameters:

• Western blotting: Select antibodies validated for this application with demonstrated specificity at ~60 kDa (observed molecular weight) .

• Immunohistochemistry: Choose antibodies specifically validated in fixed tissues with optimized antigen retrieval protocols.

• ELISA: Consider using matched antibody pairs (capture and detection) designed for ELISA applications .

Technical specifications:

• Clone type: Monoclonal antibodies offer higher specificity but may have limited epitope recognition. Recombinant antibodies provide better lot-to-lot consistency .

• Host species: Consider secondary antibody compatibility and potential cross-reactivity with your sample species.

• Storage and handling: Most antibodies require storage at -20°C, with avoidance of repeated freeze/thaw cycles .

Validation documentation: Request data showing antibody validation in your specific application and cell/tissue type .

How can researchers troubleshoot weak or absent phospho-SMAD5 signals in their experiments?

When encountering weak or absent phospho-SMAD5 signals, systematically address these potential issues:

Sample preparation issues:

• Insufficient phosphorylation induction: Verify BMP ligand activity and increase concentration (up to 30-100 ng/mL). Consider time-course experiments (5-60 minutes) .

• Rapid dephosphorylation: Add phosphatase inhibitors (25 mM NaF, 25 mM Na β-glycerophosphate) to all buffers .

• Protein degradation: Use protease inhibitor cocktail and maintain cold temperature during sample handling.

Technical issues:

• Antibody dilution: Optimize antibody concentration; try using more concentrated primary antibody (1:500-1:1000) .

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C rather than 2-3 hours .

• Detection sensitivity: Switch to more sensitive detection systems (e.g., enhanced chemiluminescence for Western blot).

Biological variables:

• Receptor expression: Confirm expression of BMP receptors (ALK2/3/6) in your cell model.

• Pathway inhibition: Test for expression of SMAD pathway inhibitors (e.g., Smurfs, NOG).

• Cell-specific differences: Some cell types may require different stimulation conditions or exhibit different baseline phosphorylation.

Validation experiment suggestions:

• Include a positive control (e.g., C2C12 cells treated with BMP4)

• Use phosphatase treatment as a negative control

• Test multiple antibody clones if possible

• Verify phosphorylation using a parallel technique (e.g., both Western blot and immunofluorescence)

How do you distinguish true phospho-SMAD5 signals from cross-reactivity with other phosphorylated SMAD proteins?

Distinguishing true phospho-SMAD5 signals from cross-reactivity requires careful experimental design:

Understanding antibody specificity:
Many commercial phospho-SMAD5 (S463/S465) antibodies cross-react with phospho-SMAD1 (S463/S465) and sometimes phospho-SMAD9 (S465/S467) due to high sequence homology in the phosphorylation regions . This is evidenced by data sheets listing cross-reactivity with multiple SMAD proteins.

Validation strategies:

• Knockout/knockdown controls: Use SMAD5-specific siRNA/shRNA while maintaining SMAD1/SMAD9 expression to determine the SMAD5-specific component of your signal.

• Recombinant protein standards: Run purified phosphorylated forms of SMAD1, SMAD5, and SMAD9 alongside your samples to create a reference pattern.

• Isoform-specific regions: Target antibodies to regions outside the phosphorylation site that differ between SMAD1, SMAD5, and SMAD9 for follow-up confirmation.

• Phosphorylation-specific inhibitors: Use ALK receptor-specific inhibitors like DMH-1 (shown to inhibit SMAD1/5 phosphorylation in a dose-dependent manner) .

Data interpretation approach:

• Report signals as "phospho-SMAD1/5" when using antibodies known to detect both

• Perform parallel experiments with total SMAD5-specific antibodies to confirm expression

• Use multiple antibody clones from different vendors and compare results

• Consider mass spectrometry-based approaches for definitive identification

What controls should be included when assessing phospho-SMAD5 levels in response to experimental manipulations?

A comprehensive set of controls is essential for accurate interpretation of phospho-SMAD5 experiments:

Essential experimental controls:

• Baseline/unstimulated control: Cells maintained in the same conditions without BMP/TGF-β stimulation to establish baseline phosphorylation .

• Total SMAD5 measurement: Parallel detection of total (non-phosphorylated + phosphorylated) SMAD5 to normalize phosphorylation signals and account for expression variations .

• Loading/housekeeping control: GAPDH, β-actin, or another stable reference protein to ensure equal protein loading across samples .

• Pathway inhibition control: Pre-treatment with specific inhibitors like DMH-1 (BMP type I receptor inhibitor) to confirm signal specificity. Figure 3 from source demonstrates dose-dependent inhibition of BMP4-induced SMAD1/5 phosphorylation by DMH-1 .

• Phosphatase treatment control: Sample aliquot treated with λ-phosphatase to confirm phosphorylation-specific detection.

Additional validation controls:

• Time course: Multiple time points (5, 15, 30, 60, 120 minutes) following stimulation to capture phosphorylation dynamics.

• Dose response: Range of BMP/TGF-β concentrations (1-100 ng/mL) to establish dose-dependent effects, as shown in Figures 1 and 2 from source .

• Secondary antibody-only control: Omission of primary antibody to assess non-specific binding of secondary antibodies.

• Known positive sample: Cell line with well-characterized SMAD5 phosphorylation (e.g., C2C12 cells treated with BMP4) .

• Environmental variable control: For studies involving pH, temperature, or osmolarity changes, include appropriate vehicle controls to isolate effects on SMAD5 phosphorylation .

How can researchers investigate the distinct cytoplasmic functions of non-phosphorylated SMAD5?

Recent research has revealed a novel, non-canonical role for cytoplasmic SMAD5 independent of its transcriptional activity . To investigate these functions:

Experimental approaches:

• Subcellular fractionation coupled with immunoblotting:

  • Separate nuclear and cytoplasmic fractions

  • Immunoblot for SMAD5 in each fraction

  • Compare phosphorylated vs. total SMAD5 distribution

• Mutant SMAD5 constructs:

  • Create phospho-mimetic mutants (S463E/S465E) that simulate constitutive phosphorylation

  • Create phospho-resistant mutants (S463A/S465A) that cannot be phosphorylated

  • Express these constructs and assess cytoplasmic functions

• Co-immunoprecipitation to identify interacting partners:

  • Target hexokinase 1 (HK1), a validated SMAD5 interactor

  • Use mass spectrometry to identify novel cytoplasmic binding partners

  • Confirm interactions through reciprocal pulldowns

• Metabolic function assessment:

  • Measure glycolytic rates in SMAD5 knockout/knockdown cells

  • Assess mitochondrial morphology and oxygen consumption

  • Analyze HK1 activity in relation to SMAD5 status

• pH sensitivity experiments:

  • Manipulate intracellular pH through media pH changes, weak acids/bases

  • Track SMAD5 localization through live-cell imaging with GFP-tagged SMAD5

  • Correlate pH changes with metabolic alterations

This research has shown that cytoplasmic SMAD5 interacts directly with HK1, enhancing glycolysis particularly under alkaline intracellular pH conditions, an interaction that becomes stronger as pHi increases .

What are the methodological approaches for studying pH-dependent nucleocytoplasmic shuttling of SMAD5?

The discovery that SMAD5 functions as an intracellular pH messenger through nucleocytoplasmic shuttling provides an exciting research area . To study this phenomenon:

Live-cell imaging approaches:

• GFP-tagged SMAD5 constructs: Generate stable cell lines expressing GFP-SMAD5 for real-time visualization of localization changes.

• Environmental manipulations:

  • Acidic conditions: Apply 10 mM hydrochloric acid treatments (induces nuclear localization)

  • Alkaline conditions: Apply 2 mM sodium hydroxide (induces cytoplasmic translocation)

  • Temperature changes: Cold (cytoplasmic) vs. heat (nuclear) treatments

  • Osmolarity: Hypotonic (nuclear) vs. hypertonic (cytoplasmic) conditions

Quantitative assessment methods:

• Nuclear/cytoplasmic fluorescence ratio: Measure GFP intensity in defined nuclear and cytoplasmic regions over time.

• High-content imaging: Automated measurement of subcellular distribution across many cells simultaneously.

• pHi measurement: Use pHi-sensitive dyes (BCECF, SNARF) concurrently with localization studies.

Mechanistic investigations:

• Mutation of charged amino acid clusters within the MH1 domain that serve as pH sensors.

• Inhibition of nuclear export machinery using compounds like leptomycin B.

• Biophysical measurements of proton dissociation from SMAD5 under varying pH conditions.

Research has demonstrated that SMAD5 responds to pHi changes within physiologically relevant ranges, with nuclear accumulation at pHi below 6.62 and cytoplasmic distribution at pHi above 7.62 .

How can researchers dissect the differential roles of phosphorylated SMAD5 in normal development versus disease states?

Investigating SMAD5 phosphorylation in developmental processes versus pathological conditions requires sophisticated approaches:

Developmental context investigation:

• Stem cell differentiation models:

  • Neural specification: Track phospho-SMAD5 during human embryonic stem cell differentiation into neural lineages

  • Hematopoietic development: Monitor erythroid progenitor proliferation in response to BMP4-induced SMAD5 phosphorylation

• Conditional knockout models:

  • Generate tissue-specific and temporally-controlled SMAD5 knockout systems

  • Introduce rescue constructs with wild-type or phospho-mutant SMAD5

  • Assess developmental outcomes and correlate with phosphorylation status

• Developmental timing analysis:

  • Create phosphorylation state-specific reporter systems

  • Map phospho-SMAD5 patterns throughout embryonic development

  • Correlate with morphological and functional changes

Disease-specific approaches:

• Patient-derived samples:

  • Compare phospho-SMAD5/total SMAD5 ratios in normal versus diseased tissues

  • Correlate phosphorylation status with disease progression

  • Develop tissue microarrays for high-throughput analysis

• Disease models:

  • Respiratory system disorders (COPD, asthma, PAH, lung cancer, IPF)

  • Cancer progression models

  • Developmental disorders associated with BMP/TGF-β signaling

• Therapeutic intervention assessment:

  • Test compounds targeting BMP receptors (e.g., DMH-1)

  • Evaluate effects on SMAD5 phosphorylation and downstream outcomes

  • Develop phosphorylation-state specific inhibitors

Research has shown that dysregulation of SMAD5 phosphorylation can have significant implications in developmental disorders and cancer progression, making it a promising therapeutic target .

What are the latest methods for multiplexed analysis of SMAD5 phosphorylation in relation to other signaling pathways?

Modern research increasingly examines SMAD5 phosphorylation within broader signaling networks:

Advanced multiplexing techniques:

• Phosphoproteomics approaches:

  • Mass spectrometry-based phosphopeptide enrichment and quantification

  • RPPA (Reverse Phase Protein Array) for high-throughput phosphorylation profiling across multiple pathways simultaneously

  • Targeted mass spectrometry using selected reaction monitoring (SRM) for specific phosphopeptides

• Multi-parameter flow cytometry:

  • Single-cell analysis of phospho-SMAD5 alongside other phosphorylated proteins

  • Correlation with cell cycle status, differentiation markers, or viability parameters

  • Cell sorting based on phosphorylation patterns for downstream analysis

• Spatial proteomics:

  • Multiplexed immunofluorescence using spectral unmixing

  • Imaging mass cytometry for high-parameter tissue analysis

  • Digital spatial profiling technologies

Integrated pathway analysis:

• Combinatorial perturbation experiments:

  • Simultaneous modulation of BMP/TGF-β with intersecting pathways (MAPK, PI3K/AKT)

  • Compound effect assessment using phosphorylation readouts

  • Mathematical modeling of signaling cross-talk

• Temporal dynamics investigation:

  • Kinetic measurements of phosphorylation events using time-course experiments

  • Pulse-chase approaches to determine phosphorylation stability

  • Live-cell imaging with phosphorylation-specific biosensors

Research using these approaches has revealed that phospho-SMAD5 signaling integrates with other pathways in complex ways, such as during viral infections when multiple signaling cascades activate simultaneously , providing a more comprehensive understanding of cellular response mechanisms.