Phospho-APLF (S116) Antibody

What is APLF and why is its phosphorylation at Ser116 significant?

APLF (Aprataxin and PNK-like Factor) is a multifunctional protein involved in the DNA damage response (DDR). It contains a forkhead-associated (FHA) domain, Ku-binding motif (KBM), poly(ADP-ribose)-binding zinc finger (PBZ) domains, and an acidic domain (AD). APLF undergoes ionizing radiation (IR)-induced and Ataxia-Telangiectasia Mutated (ATM)-dependent phosphorylation at serine-116 (Ser116) .

The phosphorylation at Ser116 is particularly significant because it:

• Enhances APLF retention at DNA damage sites

• Facilitates efficient DNA double-strand break (DSB) repair kinetics

• Contributes to cell survival following ionizing radiation

• Appears to be required for APLF-dependent non-homologous end joining (NHEJ)

How does the PARP3-ATM signaling pathway regulate APLF phosphorylation?

Research has revealed that APLF phosphorylation involves a coordinated pathway:

• Following DNA damage, APLF is initially recruited to damage sites through its PBZ domains' interaction with poly(ADP-ribose) (PAR) chains

• PARP3 (not PARP1 or PARP2) is specifically required for APLF Ser116 phosphorylation

• ATM kinase directly phosphorylates APLF at Ser116

• The process forms a feedback loop where:

  • PAR-dependent interaction brings APLF to DNA damage sites

  • PARP3 facilitates ATM-dependent phosphorylation of APLF

  • Phosphorylated APLF shows enhanced retention at damage sites

This pathway represents a novel signaling mechanism specific to DSB repair, distinct from single-strand break response mechanisms.

What are the optimal applications and dilutions for Phospho-APLF (S116) Antibody?

The antibody detects endogenous levels of APLF protein only when phosphorylated at S116, making it ideal for studying DNA damage response dynamics in fixed cells and tissues .

What are the essential controls for validating Phospho-APLF (S116) Antibody specificity?

To ensure reliable results, include these critical controls:

• Negative controls:

  • APLF-depleted cells (siRNA or shRNA knockdown)

  • APLF S116A mutant (serine to alanine substitution prevents phosphorylation)

  • Non-irradiated cells (minimal basal phosphorylation)

• Positive controls:

  • Cells treated with DNA-damaging agents known to activate ATM:

    • Ionizing radiation (IR)

    • Hydrogen peroxide (H₂O₂)

    • Camptothecin (CPT)

    • Etoposide (VP16)

    • Methyl methanesulfonate

• Specificity controls:

  • Pre-absorption with immunogen peptide

  • ATM inhibitor treatment (KU55933) should abolish signal

  • UV-C radiation (activates ATR but not ATM) should not induce signal

These controls help distinguish true phospho-S116 signal from background or cross-reactivity.

How should DNA damage induction be optimized for phospho-APLF studies?

For robust and reproducible phospho-APLF (S116) detection:

• Optimal DNA damage induction:

  • Ionizing radiation: 2-10 Gy is sufficient (phosphorylation detectable at 1 min, peaks within first hour)

  • Chemical agents: Use etoposide (10 μM), hydrogen peroxide (100 μM), or camptothecin (1 μM)

  • Avoid UV-C radiation or hydroxyurea, which don't efficiently induce APLF-S116 phosphorylation

• Kinetics considerations:

  • Early timepoints (1-60 min) capture peak phosphorylation

  • Signal returns to baseline by 24 hours post-irradiation

  • Include multiple timepoints when studying repair dynamics

• Cell type considerations:

  • U2OS cells show robust phosphorylation response

  • HEK293T cells may show some basal phosphorylation, especially with overexpressed APLF

What factors can affect antibody performance in phospho-APLF detection?

Several experimental factors can significantly impact results:

• Fixation methods:

  • Paraformaldehyde fixation preserves phospho-epitopes

  • Avoid methanol fixation which can remove phosphorylations

  • For tissue sections, antigen retrieval using high-pressure and temperature Tris-EDTA (pH 8.0) is recommended

• Technical considerations:

  • Include phosphatase inhibitors in all buffers during cell/tissue processing

  • Fresh antibody dilutions perform better than stored dilutions

  • Optimize incubation time (4°C overnight often yields best results for IHC)

• Signal detection issues:

  • Excessive antibody concentration may increase background

  • Insufficient washing can leave non-specific binding

  • Signal may be focal/punctate rather than diffuse when examining IR-induced damage sites

How can phospho-APLF (S116) antibody be used to study DNA repair pathway dynamics?

Phospho-APLF antibody enables several sophisticated experimental approaches:

• Laser microirradiation studies:

  • Track real-time recruitment and retention of phosphorylated APLF at DNA damage sites

  • Compare wild-type vs. S116A/S116D mutants in live-cell imaging

  • Quantify the kinetics of phospho-APLF accumulation (rapid recruitment followed by ATM-dependent retention)

• Chromatin fractionation assays:

  • Isolate chromatin-bound proteins at various timepoints after damage

  • Quantify phospho-APLF levels in different cellular fractions

  • Determine how APLF phosphorylation affects recruitment of other repair factors like XRCC4

• Immunofluorescence co-localization:

  • Examine co-localization of phospho-APLF with:

    • γH2AX foci (DSB marker)

    • PARP3

    • Other repair factors

  • Use to study spatiotemporal dynamics of repair complex assembly

What approaches can resolve contradictory findings when using phospho-APLF antibody?

When encountering inconsistent results:

• DNA damage type considerations:

  • Different damage types induce varied phosphorylation patterns

  • IR and radiomimetic drugs activate primarily ATM-dependent pathways

  • Replication stress activates primarily ATR pathways (minimal APLF-S116 phosphorylation)

• PARP inhibitor effects:

  • Different PARP inhibitors have varying selectivity for PARP3

  • AZD2281 significantly impairs APLF-S116 phosphorylation

  • PJ-34 or ABT888 have minimal effect on phosphorylation

  • Consider isoform specificity when interpreting inhibitor results

• Cell cycle considerations:

  • Cell cycle phase may affect phosphorylation efficiency

  • Use cell synchronization or cell cycle markers to resolve discrepancies

  • Non-dividing (confluent) cells may have different repair pathway utilization

How can phospho-APLF antibody be used to investigate the functional relationship between APLF's histone chaperone activity and its phosphorylation?

Recent research has revealed APLF's role as a histone chaperone, opening new research avenues:

• Chromatin assembly assays:

  • Compare histone chaperone activity of wild-type vs. phospho-mimetic (S116D) vs. phospho-deficient (S116A) APLF

  • Use supercoiling assays with purified human histones and topoisomerase I

  • Evaluate ATP-dependent chromatin assembly with CHD1 remodeler

• Histone interaction studies:

  • Investigate whether Ser116 phosphorylation affects APLF's interaction with H3/H4 tetramers

  • Perform salt-gradient immunoprecipitations to assess stability of histone interactions

  • Determine if phosphorylation alters binding specificity for histone variants

• Nucleosome dynamics at damage sites:

  • Track histone displacement/incorporation at damage sites with fluorescently-tagged histones

  • Examine how APLF phosphorylation affects chromatin relaxation during repair

  • Investigate recruitment of histone variant macroH2A1 in relation to APLF phosphorylation status

What is the correlation between APLF Ser116 phosphorylation and clinical outcomes in cancer research?

Recent findings suggest important clinical implications:

• Cisplatin resistance:

  • Cisplatin-resistant cancer cells show elevated APLF levels

  • APLF depletion sensitizes cells to cisplatin treatment

  • APLF facilitates interstrand crosslink (ICL) repair and replication fork protection

• Research approaches:

  • Use phospho-APLF antibody to assess phosphorylation status in patient-derived samples

  • Correlate APLF phosphorylation with treatment response

  • Examine phospho-APLF as a potential biomarker for DNA damage response proficiency

• Therapeutic implications:

  • APLF phosphorylation status may predict DNA repair capacity

  • Combined targeting of APLF and ATM pathways could enhance chemotherapy efficacy

  • Monitor phospho-APLF levels as a pharmacodynamic marker during treatment

How does APLF Ser116 phosphorylation impact DNA repair pathway choice?

Research findings reveal complex regulatory mechanisms:

• NHEJ efficiency:

  • APLF S116A mutation reduces random plasmid integration efficiency by approximately 40%

  • Phospho-mimetic APLF S116D rescues NHEJ efficiency to near wild-type levels

  • Phosphorylation affects the stability of XRCC4 at damaged chromatin

• γH2AX resolution kinetics:

  • Cells expressing APLF S116A show significantly more residual γH2AX foci at 4 and 24 hours post-irradiation

  • This indicates delayed DSB repair when APLF cannot be phosphorylated

  • The effect is comparable to APLF depletion, suggesting phosphorylation is critical for function

• Cellular radiosensitivity:

  • Clonogenic survival assays show increased radiosensitivity with APLF S116A expression

  • This confirms the biological significance of phosphorylation for cell survival

  • The data collectively suggest phosphorylation enhances APLF-dependent repair pathway efficiency

What methodological approaches can resolve temporal dynamics of APLF phosphorylation in the DNA damage response?

To capture the complete timeline of APLF function:

• High-resolution temporal analysis:

  • Phospho-APLF is detectable as early as 1 minute post-irradiation

  • Signal peaks within the first hour and returns to pre-irradiation levels by 24 hours

  • Use time-course experiments with multiple early timepoints (1, 5, 15, 30, 60 minutes)

• Live-cell imaging approaches:

  • Combine fluorescently-tagged APLF with phospho-specific antibody staining

  • Track recruitment, phosphorylation, and retention phases separately

  • Use FRAP (Fluorescence Recovery After Photobleaching) to assess kinetics of phospho-APLF at damage sites

• Multi-modal analysis:

  • Correlate antibody-based detection with functional assays at matched timepoints

  • Track DSB repair progression using surrogate markers (γH2AX, 53BP1)

  • Integrate data to create comprehensive models of APLF's phosphorylation-dependent functions in repair