Phospho-ATF2 (T71) Recombinant Monoclonal Antibody

What is Phospho-ATF2 (T71) and why is it important in cellular signaling?

Phospho-ATF2 (T71) refers to the Activating Transcription Factor 2 (ATF2) protein that has been phosphorylated at threonine 71. ATF2 functions as a critical transcriptional activator that regulates the expression of various genes involved in anti-apoptosis, cell growth, and DNA damage response pathways . The phosphorylation at threonine 71 is a key post-translational modification that activates ATF2, enabling it to perform its diverse cellular functions. This phosphorylation is mediated by several kinases, including stress-activated protein kinases (SAPKs), p38, and JNK . The importance of this phosphorylation lies in its role as a molecular switch that alters ATF2's activity, subcellular localization, and interaction partners. In particular, the phosphorylated form (mediated by ATM) plays a crucial role in the DNA damage response and is involved in ionizing radiation-induced S phase checkpoint control . Understanding ATF2 phosphorylation status provides insights into cellular responses to stress and various signaling pathways that regulate cell survival, proliferation, and death.

How does phosphorylated ATF2 differ functionally from non-phosphorylated ATF2?

Phosphorylation of ATF2 at threonine 71 (and often in conjunction with threonine 69) creates significant functional differences compared to its non-phosphorylated form. When phosphorylated, ATF2 exhibits enhanced DNA binding affinity to specific consensus sequences, including CRE (cAMP response element) sequences (5'-TGACGTCA-3') and AP-1 (activator protein 1) sequences (5'-TGACTCA-3') . This phosphorylation-dependent binding allows for selective gene transcription regulation.

The functional differences include:

Additionally, phosphorylated ATF2 has been shown to facilitate the recruitment of the MRN complex into ionizing radiation-induced foci (IRIF), a critical step in DNA damage repair processes . Research has also demonstrated that the phosphorylation of ATF2 can promote viral replication, as evidenced in studies with pseudorabies virus (PRV), where increased phosphorylation levels of ATF2 enhanced viral replication efficiency .

What are the optimal conditions for Western Blot applications using Phospho-ATF2 (T71) antibody?

Recommended Western Blot protocol elements:

• Sample preparation:

  • Extract proteins under phosphatase inhibitor protection

  • Use fresh samples when possible to minimize phospho-epitope degradation

  • Include positive controls (cells treated with stress inducers like UV or anisomycin)

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Load 20-40 μg of total protein per lane

  • Include molecular weight markers (ATF2 appears at approximately 70 kDa)

• Transfer and blocking:

  • Transfer to PVDF membrane (preferred over nitrocellulose for phospho-epitopes)

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

  • Incubate with primary antibody overnight at 4°C

• Detection:

  • Use HRP-conjugated secondary antibody (typically anti-rabbit IgG)

  • Consider enhanced chemiluminescence detection systems

  • Validate specificity using phosphatase-treated control samples

The antibody is most effective when samples are freshly prepared and stored appropriately, as repeated freeze-thaw cycles should be avoided to maintain phospho-epitope integrity .

How can immunofluorescence protocols be optimized for Phospho-ATF2 (T71) detection?

For immunofluorescence (IF) applications, optimization of several parameters is crucial to achieve specific and sensitive detection of Phospho-ATF2 (T71). The recommended dilution range for IF applications is 1:20-1:200 , though this should be empirically determined for each experimental system.

Detailed immunofluorescence optimization strategy:

• Fixation and permeabilization:

  • Test both paraformaldehyde (4%) and methanol fixation methods

  • For paraformaldehyde fixation, permeabilize with 0.1-0.5% Triton X-100

  • Methanol fixation often preserves phospho-epitopes better but test both

• Blocking conditions:

  • Use 5-10% normal serum (different from antibody host species) with 1% BSA

  • Include 0.1% Triton X-100 in blocking buffer for nuclear antigens

  • Block for minimum 1 hour at room temperature

• Antibody incubation:

  • Start with 1:50 dilution and optimize

  • Incubate primary antibody overnight at 4°C

  • Use gentle rocking/rotation during incubation

• Signal enhancement strategies:

  • Consider tyramide signal amplification for low abundance targets

  • Use high-sensitivity fluorophore-conjugated secondary antibodies

  • Include DAPI counterstain for nuclear visualization (ATF2 has nuclear localization)

• Controls:

  • Phosphatase-treated negative control

  • Stimulated cell positive control (e.g., UV radiation, JNK activators)

  • Secondary antibody-only control

This antibody is compatible with fluorescent detection systems and can be used to visualize the subcellular localization of phosphorylated ATF2, which is predominantly nuclear when activated .

What controls should be included when validating Phospho-ATF2 (T71) antibody specificity?

Rigorous validation of antibody specificity is essential for reliable research results, particularly for phospho-specific antibodies. For Phospho-ATF2 (T71) antibody, several controls should be implemented:

Additionally, researchers should consider:

• Testing the antibody across multiple techniques (WB, IF, ELISA) to confirm consistent specificity

• Using stimulation time-course experiments to correlate signal with known ATF2 phosphorylation kinetics

• Comparing results with alternative phospho-ATF2 (T71) antibodies from different suppliers or clones

• When possible, implementing mass spectrometry validation of the phosphorylation site in immunoprecipitated samples

For advanced validation, consider using cells expressing ATF2 with T71A mutation (preventing phosphorylation) as a definitive negative control, though this requires special preparation and is not commercially available .

How can Phospho-ATF2 (T71) antibody be used to study DNA damage response pathways?

Phospho-ATF2 (T71) antibody serves as a valuable tool for investigating DNA damage response (DDR) pathways, given ATF2's significant role in these processes. The phosphorylated form of ATF2 (mediated by ATM) is specifically involved in the ionizing radiation (IR)-induced S phase checkpoint control and in recruiting the MRN complex to IR-induced foci (IRIF) . Researchers can leverage this antibody to explore multiple aspects of the DDR pathway:

• Temporal dynamics of ATF2 activation:

  • Treat cells with DNA damaging agents (ionizing radiation, genotoxic chemicals)

  • Harvest at various time points (5 min to 24 hours)

  • Quantify phospho-ATF2 levels via Western blot or flow cytometry

  • Correlate with activation of upstream kinases (ATM, JNK, p38)

• Spatial organization of DDR components:

  • Perform co-immunofluorescence with phospho-ATF2 (T71) antibody and other DDR proteins

  • Visualize recruitment to damage foci using confocal microscopy

  • Quantify colocalization with MRN complex components (MRE11, RAD50, NBS1)

  • Analyze nuclear redistribution patterns during DDR activation

• Pathway interaction studies:

  • Combine with inhibitors of ATM, ATR, DNA-PK, JNK, or p38 pathways

  • Assess changes in ATF2 phosphorylation status

  • Determine pathway dependencies for different damage types

  • Analyze feedback mechanisms between ATF2 and other DDR components

• Chromatin immunoprecipitation (ChIP) applications:

  • Use phospho-ATF2 (T71) antibody for ChIP experiments

  • Map binding sites on damage-responsive genes

  • Quantify recruitment to specific genomic loci following damage

  • Compare binding patterns between different damage types

This antibody enables researchers to discern the specific roles of phosphorylated ATF2 versus total ATF2 protein in DDR processes, providing insights into the molecular mechanisms underlying genome stability maintenance .

What is the relationship between ATF2 phosphorylation and viral replication, and how can it be studied?

The relationship between ATF2 phosphorylation and viral replication represents an emerging area of research with significant implications for understanding host-pathogen interactions. Studies have demonstrated that ATF2 phosphorylation plays a critical role in promoting viral replication, particularly for viruses like pseudorabies virus (PRV) . This relationship can be systematically investigated using Phospho-ATF2 (T71) antibody through several experimental approaches:

• Viral infection models:

  • Infect appropriate cell lines with virus of interest

  • Monitor changes in ATF2 phosphorylation status over the course of infection

  • Correlate phosphorylation with viral replication markers

  • Compare responses across different viral strains and multiplicities of infection

• Mechanistic investigation:

  • Implement RNAi knockdown of ATF2 or CRISPR/Cas9 gene knockout as performed in previous studies

  • Assess impact on viral titer and viral genome copy number

  • Rescue experiments with wild-type vs. phospho-mutant ATF2 (T69A/T71A)

  • Determine whether viral proteins directly interact with ATF2

• Signaling pathway analysis:

  • Use inhibitors of JNK pathway components (demonstrated to mediate ATF2 activation during infection)

  • Monitor effects on both ATF2 phosphorylation and viral replication

  • Identify viral proteins that may activate kinases upstream of ATF2

  • Perform phosphoproteomics to identify additional targets in the pathway

• Transcriptional regulation studies:

  • Use ChIP-seq with phospho-ATF2 (T71) antibody in infected vs. uninfected cells

  • Identify viral and host genes regulated by phosphorylated ATF2

  • Analyze promoter regions for ATF2 binding motifs (CRE or AP-1 sites)

  • Validate findings with reporter assays and site-directed mutagenesis

Research has specifically shown that ATF2-knockdown reduces both PRV titer and viral genome copy number, while overexpression of ATF2 and phosphorylation at positions 69/71 promotes PRV replication . These findings suggest that targeting ATF2 phosphorylation could provide a potential therapeutic strategy for inhibiting viral infections.

How can researchers distinguish between the nuclear and cytoplasmic functions of phosphorylated ATF2?

ATF2 exhibits distinct functions depending on its subcellular localization. In the nucleus, it contributes to transcriptional regulation and DNA damage response, while in the cytoplasm, it interacts with mitochondrial membrane proteins like HK1 and VDAC1 to influence mitochondrial membrane potential and cell death . Distinguishing between these compartment-specific functions requires sophisticated experimental approaches:

• Subcellular fractionation combined with Western blotting:

  • Perform careful subcellular fractionation to separate nuclear, cytoplasmic, and mitochondrial fractions

  • Use Phospho-ATF2 (T71) antibody to quantify distribution across compartments

  • Validate fraction purity with compartment-specific markers (e.g., Lamin B for nucleus, GAPDH for cytoplasm)

  • Compare phosphorylation status across compartments

• High-resolution microscopy approaches:

  • Implement super-resolution microscopy techniques (STORM, PALM, or SIM)

  • Perform multi-color immunofluorescence with Phospho-ATF2 (T71) antibody (1:20-1:200 dilution)

  • Co-localize with compartment markers and functional partners

  • Quantify distribution changes following various cellular stimuli

• Experimental manipulation of localization:

  • Generate ATF2 constructs with mutated nuclear localization signals or nuclear export signals

  • Create fusion proteins with compartment-targeting sequences

  • Compare phosphorylation-dependent activities in different compartments

  • Implement inducible translocation systems for temporal control

• Proximity-based labeling methods:

  • Implement BioID or APEX2 proximity labeling fused to ATF2

  • Identify compartment-specific interaction partners of phosphorylated ATF2

  • Compare interactomes between wild-type and phospho-mutant versions

  • Validate key interactions with co-immunoprecipitation using Phospho-ATF2 (T71) antibody

• Compartment-specific functional assays:

  • For nuclear functions: ChIP-seq, transcriptional reporter assays

  • For cytoplasmic/mitochondrial functions: membrane potential assays, cytochrome c release, interaction with HK1/VDAC1

These approaches, when used in combination, allow researchers to parse the complex dual functionality of phosphorylated ATF2 and determine how phosphorylation at T71 impacts its compartment-specific roles in both normal cellular processes and disease states .

How should researchers quantify and normalize Phospho-ATF2 (T71) signals in Western blot experiments?

Accurate quantification and normalization of Phospho-ATF2 (T71) signals in Western blot experiments are essential for reliable interpretation of results. This process requires careful attention to several methodological considerations:

• Image acquisition parameters:

  • Use a wide dynamic range detection system (digital imaging preferred over film)

  • Ensure exposure is within linear range (no saturated pixels)

  • Maintain consistent acquisition settings across experimental replicates

  • Capture both phospho-ATF2 and normalization controls in same image when possible

• Quantification approach:

  • Use dedicated image analysis software (ImageJ, Image Lab, etc.)

  • Perform densitometry on defined regions of interest

  • Subtract local background for each band

  • Generate integrated density values (area × mean intensity)

• Normalization strategies:

• Statistical analysis:

  • Perform experiments in biological triplicate (minimum)

  • Apply appropriate statistical tests (t-test, ANOVA) based on experimental design

  • Report both fold changes and p-values

  • Present data with error bars representing standard deviation or standard error

• Special considerations for phospho-epitopes:

  • Always include positive controls (cells treated with known activators)

  • Consider phosphorylation kinetics (time-course experiments)

  • Be aware of potential rapid dephosphorylation during sample preparation

  • Use phosphatase inhibitors consistently

When working with the Phospho-ATF2 (T71) antibody at the recommended dilutions (1:500-1:5000) , researchers should optimize exposure times to ensure signals fall within the linear detection range of their imaging system, which is critical for accurate quantification of subtle changes in phosphorylation status.

What approaches can be used to study the interplay between ATF2 phosphorylation at T71 and other post-translational modifications?

ATF2 function is regulated by a complex network of post-translational modifications (PTMs), with phosphorylation at T71 being a critical regulatory site that often works in concert with other modifications. Understanding this interplay requires specialized experimental approaches:

• Sequential and combinatorial phosphorylation analysis:

  • Investigate the relationship between T71 phosphorylation and T69 phosphorylation

  • Perform time-course experiments with various stimuli

  • Use site-specific phospho-antibodies (including Phospho-ATF2 (T71) antibody)

  • Implement phospho-mimetic and phospho-resistant mutations (T71E/D vs. T71A)

• Cross-talk with other modification types:

  • Examine interactions between phosphorylation and other PTMs (acetylation, SUMOylation, ubiquitination)

  • Use deacetylase inhibitors, proteasome inhibitors, or SUMO pathway modulators

  • Implement mass spectrometry-based approaches for global PTM mapping

  • Develop or acquire modification-specific antibodies for co-detection studies

• PTM-dependent protein interactions:

  • Perform co-immunoprecipitation with Phospho-ATF2 (T71) antibody

  • Compare interactome of phosphorylated vs. non-phosphorylated ATF2

  • Identify readers of the phospho-T71 mark

  • Implement proximity ligation assays to detect interactions in situ

• Functional outcomes of modification patterns:

  • Assess DNA binding properties using electrophoretic mobility shift assays

  • Measure transcriptional activity with reporter constructs

  • Evaluate histone acetyltransferase (HAT) activity with in vitro assays

  • Determine effects on cellular processes (DNA damage response, apoptosis, cell cycle)

• Pathway integration analysis:

  • Map kinase signaling pathways leading to different modification patterns

  • Use specific inhibitors of JNK, p38, ATM, and other relevant kinases

  • Implement mathematical modeling to predict modification interdependencies

  • Correlate phosphorylation patterns with biological outcomes

These approaches enable researchers to move beyond single-modification studies and develop a more comprehensive understanding of how T71 phosphorylation coordinates with other modifications to fine-tune ATF2 function in different cellular contexts and in response to various stimuli .

How can researchers resolve contradictory data regarding ATF2 phosphorylation across different experimental systems?

Contradictory findings regarding ATF2 phosphorylation across different experimental systems are not uncommon and can result from various methodological, biological, and technical factors. Resolving such discrepancies requires systematic troubleshooting and careful experimental design:

• Methodological standardization:

  • Compare antibody performance across studies (clone, supplier, lot number)

  • Standardize application protocols (dilutions, incubation conditions)

  • Implement consistent sample preparation methods

  • Use recombinant phosphorylated standards for cross-laboratory calibration

• Biological system variations:

  • Analyze cell type-specific effects (ATF2 can elicit oncogenic or tumor suppressor activities depending on tissue/cell type)

  • Consider species differences in ATF2 sequence and regulation

  • Evaluate the impact of culture conditions and passage number

  • Assess microenvironmental factors that might influence signaling

• Technical approach diversification:

  • Validate findings across multiple techniques (WB, IF, flow cytometry, ELISA)

  • Implement complementary approaches (mass spectrometry for direct PTM detection)

  • Use genetic approaches (CRISPR/Cas9 editing, site-directed mutagenesis)

  • Conduct functional validation experiments

• Systematic meta-analysis framework:

• Collaborative validation approaches:

  • Implement multi-laboratory testing of identical samples

  • Share positive control materials and protocols

  • Develop consensus guidelines for ATF2 phosphorylation analysis

  • Create repository of validated reagents and protocols

When working with Phospho-ATF2 (T71) antibody specifically, researchers should ensure they are using validated antibodies such as the recombinant rabbit monoclonal antibody (clone 4F2) at appropriate dilutions for each application (WB:1:500-1:5000, IF:1:20-1:200) , while implementing appropriate controls for each experimental system to facilitate accurate cross-study comparisons.

How can Phospho-ATF2 (T71) antibody be used in studying the role of ATF2 in cancer biology?

ATF2 exhibits context-dependent roles in cancer biology, functioning as either an oncogene or tumor suppressor depending on the tissue or cell type . Phospho-ATF2 (T71) antibody provides a valuable tool for investigating these complex roles across various cancer types and experimental models:

• Cancer type-specific phosphorylation patterns:

  • Compare phospho-ATF2 levels across cancer cell lines and patient samples

  • Correlate with clinical outcomes and tumor characteristics

  • Develop tissue microarray analysis protocols using the antibody (typically at 1:50-1:100 dilution)

  • Establish prognostic significance of T71 phosphorylation in specific cancer types

• Functional studies in cancer models:

  • Manipulate ATF2 phosphorylation through kinase modulation or mutation

  • Assess impacts on hallmark cancer phenotypes (proliferation, invasion, metastasis)

  • Implement xenograft models with phosphorylation-dependent ATF2 variants

  • Correlate phosphorylation status with therapy response

• Mechanistic investigation of dual functionality:

  • Determine phosphorylation-dependent target gene specificity in cancer cells

  • Investigate compartment-specific functions (nuclear vs. cytoplasmic)

  • Analyze interaction with oncogenic and tumor suppressor pathways

  • Map cross-talk with other transcription factors and signaling molecules

• Therapeutic targeting opportunities:

  • Screen for compounds that modulate ATF2 phosphorylation

  • Assess synergistic effects with established cancer therapies

  • Develop phosphorylation-specific intervention strategies

  • Identify biomarkers for phosphorylation-dependent therapeutic responses

• Single-cell analysis approaches:

  • Implement phospho-flow cytometry with the APC-conjugated antibody

  • Analyze intratumoral heterogeneity in phosphorylation status

  • Correlate with other cancer-relevant markers

  • Track dynamic changes during tumor progression and treatment

The antibody's ability to specifically detect the phosphorylated form enables researchers to distinguish active ATF2 from total protein, providing crucial insights into its activation state in cancer cells. This is particularly important given ATF2's involvement in key cancer-related processes, including cell growth regulation, DNA damage response, and apoptosis pathways .

What are the methodological considerations for studying ATF2 phosphorylation dynamics in response to cellular stress?

Investigating the dynamic phosphorylation of ATF2 in response to cellular stress requires careful methodological considerations to capture the often rapid and transient nature of these signaling events:

• Temporal resolution optimization:

  • Design fine-grained time-course experiments (seconds to hours)

  • Implement rapid sample collection and preservation techniques

  • Use phosphatase inhibitors consistently across all samples

  • Consider live-cell imaging with phospho-specific biosensors as complementary approach

• Stress stimulus parameters:

  • Calibrate stimulus intensity to avoid oversaturation of response

  • Compare different stress types (oxidative, genotoxic, inflammatory, metabolic)

  • Implement dose-response studies alongside time-course experiments

  • Consider physiologically relevant stress conditions alongside standard laboratory stimuli

• Single-cell vs. population analysis:

  • Use flow cytometry with phospho-specific antibodies for heterogeneity assessment

  • Implement immunofluorescence microscopy for spatial information

  • Consider single-cell Western techniques for protein-limited samples

  • Correlate with single-cell transcriptomics when possible

• Pathway crosstalk analysis:

  • Simultaneously monitor multiple phosphorylation sites (T69, T71)

  • Track activation of upstream kinases (JNK, p38, ATM)

  • Use specific pathway inhibitors to dissect contribution to ATF2 phosphorylation

  • Implement systems biology approaches to model signaling networks

• Technical considerations for Phospho-ATF2 (T71) antibody use:

  • Optimize antibody concentration based on signal intensity and specificity

  • For Western blotting: use 1:500-1:5000 dilution

  • For immunofluorescence: use 1:20-1:200 dilution

  • Include both phosphorylation-positive and negative controls in each experiment

  • Consider multiple detection methods (colorimetric, chemiluminescent, fluorescent)

• Quantification and normalization approaches:

By carefully implementing these methodological considerations, researchers can achieve high-quality, reproducible data on the dynamics of ATF2 phosphorylation in response to various cellular stressors, providing insights into the temporal regulation of stress response pathways and their dysregulation in disease states .