PIAS4 Antibody

What is PIAS4 and why is it significant in molecular research?

PIAS4, also known as PIASy, PIASG, or E3 SUMO-protein ligase PIAS4, belongs to the PIAS family of proteins that function as transcriptional coregulators in various cellular pathways, including STAT signaling, p53 pathway, and steroid hormone signaling . PIAS4 primarily functions as an E3 SUMO ligase, facilitating the conjugation of small ubiquitin-like modifier (SUMO) proteins to target substrates .

Recent research has revealed PIAS4's significance in:

• Intrinsic antiviral immunity against HSV-1 infection, representing a novel aspect of PIAS protein function

• Hypoxia signaling regulation through VHL suppression in pancreatic cancer

• DNA damage response pathways by mediating sumoylation of proteins like PARP1 and KAT5

These diverse functions make PIAS4 an important research target for understanding fundamental cellular processes and disease mechanisms.

The choice between monoclonal and polyclonal PIAS4 antibodies depends on the specific research objectives:

Polyclonal PIAS4 antibodies (such as those from Proteintech and Rockland):

• Recognize multiple epitopes, providing stronger signals in applications like WB and IHC

• Show broader species cross-reactivity (human, mouse, rat)

• Useful for detecting native proteins in various applications

• Better for detecting proteins with post-translational modifications or when protein conformation may vary

Monoclonal PIAS4 antibodies (such as Cell Signaling's D2F12):

• Provide superior lot-to-lot consistency and specificity

• Often preferred for quantitative applications requiring reproducible results

• The D2F12 antibody specifically recognizes regions surrounding Lys59 of human PIAS4

• Particularly useful when specific isoforms or domains need to be distinguished

For exploratory research or when dealing with samples where PIAS4 may be modified or present at low levels, polyclonal antibodies may provide better detection. For standardized assays or when absolute specificity is required, monoclonal antibodies are preferable.

What are the optimal sample preparation protocols for detecting PIAS4 in different cellular compartments?

PIAS4 localizes to both nuclear and cytoplasmic compartments, with its distribution changing depending on cellular conditions and stimuli. Effective sample preparation is crucial for accurate detection:

Nuclear fraction enrichment:

• Use specialized nuclear extraction buffers containing 10-20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, and 10% glycerol

• Include SUMO protease inhibitors (20 mM N-ethylmaleimide) to preserve sumoylated forms

• Add phosphatase inhibitors when studying PIAS4's roles in signaling pathways

• Following HSV-1 infection, modified extraction techniques may be necessary to capture PIAS4 in viral replication compartments

Cytoplasmic detection:

• Use lower salt buffers (150 mM NaCl) supplemented with 0.5% NP-40 or Triton X-100

• When studying PIAS4's interaction with membrane-associated proteins, consider digitonin-based fractionation

Whole cell lysates:

• RIPA buffer supplementation with 1% SDS can improve detection of PIAS4, which often appears at 57-70 kDa

• Sonication is recommended to shear DNA and release chromatin-bound PIAS4

When analyzing PIAS4 in pancreatic cancer samples, researchers should note that detection can be improved using specialized extraction methods, as PIAS4 expression is significantly elevated in these tissues compared to normal pancreatic epithelium .

How should controls be designed for PIAS4 antibody validation experiments?

Rigorous validation of PIAS4 antibodies requires multiple controls:

Positive controls:

• Verified cell lines with confirmed PIAS4 expression (K-562, HL-60, Panc0327, and Panc1005 cells)

• Recombinant PIAS4 protein (full-length or the specific domain to which the antibody was raised)

• Mouse ovary or pancreas tissue, which show reliable PIAS4 expression

Negative controls:

• PIAS4 knockdown/knockout samples using validated siRNAs targeting exon 2 or exon 6

• Primary cells with naturally low PIAS4 expression (such as AsPc1 and BxPc3)

• Peptide competition assays using the immunizing peptide to confirm binding specificity

Specificity controls:

• Cross-reactivity testing with other PIAS family members (PIAS1, PIAS2, PIAS3) to ensure specificity

• Secondary antibody-only controls to rule out non-specific binding

• Isotype controls matched to the host species and antibody class

In published studies, siRNA-mediated knockdown has been particularly effective for validating PIAS4 antibody specificity, with researchers successfully targeting PIAS4 using both pooled siRNAs and individual siRNAs against specific exons .

What are the recommended protocols for immunofluorescence detection of PIAS4 in different cellular contexts?

Optimal immunofluorescence protocols for PIAS4 detection vary depending on the cellular context and research question:

Standard cultured cells (e.g., HeLa):

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 for 10 minutes

• Block with 5% BSA or 10% serum for 1 hour

• Incubate with primary antibody at dilutions of 1:50-1:500 overnight at 4°C

• Use appropriate fluorophore-conjugated secondary antibodies (1:500-1:1000)

• Counterstain nuclei with DAPI to visualize nuclear localization

Virus-infected cells (e.g., HSV-1 infection):

• Fix with 1.8% formaldehyde in PBS containing 2% sucrose

• Permeabilize with 0.5% NP-40, 10% sucrose in PBS

• For detecting PIAS4 at sites of viral genome entry, image acquisition within 1-2 hours post-infection is critical

• For replication compartment localization, imaging at 4-6 hours post-infection is recommended

Tissue sections:

• Use heat-induced epitope retrieval (citrate buffer pH 6.0)

• Longer primary antibody incubation (overnight at 4°C)

• For pancreatic tissue, background reduction with 0.3% hydrogen peroxide is recommended

• When co-staining with markers like HIF1α, sequential staining protocols may be necessary

For all applications, including a detergent in wash buffers (0.05% Tween-20) helps reduce background. When studying PIAS4's SUMO E3 ligase activity, co-staining with SUMO1/2/3 antibodies can provide valuable functional information.

How can researchers effectively use PIAS4 antibodies to study its role in hypoxia signaling pathways?

PIAS4 plays a crucial role in hypoxia signaling through its interaction with VHL (von Hippel-Lindau) protein and subsequent impact on HIF1α, making it particularly relevant in cancer research . To effectively study this pathway:

Immunoprecipitation approach:

• Use anti-PIAS4 antibodies (1:100 dilution) for immunoprecipitation from cell lysates

• Co-immunoprecipitate with anti-VHL antibodies to detect direct interaction

• Probe for sumoylated forms of VHL using anti-SUMO2/3 antibodies

• Compare normoxic and hypoxic conditions (1% O₂, 48 hours) to detect differential interactions

Chromatin immunoprecipitation (ChIP):

• Use PIAS4 antibodies to identify genomic regions where PIAS4 functions as a transcriptional coregulator

• Cross-link proteins to DNA using 1% formaldehyde for 10 minutes

• Sonicate to generate 200-500 bp DNA fragments

• Immunoprecipitate with 5 μg of anti-PIAS4 antibody

• Analyze enrichment at HIF1α target genes like VEGF and JMJD1A by qPCR

In published studies, researchers have demonstrated that PIAS4 siRNA suppresses the expression of HIF1α in pancreatic cancer cells, and when these cells were exposed to hypoxia for 48 hours, the induction of HIF1α downstream targets (VEGF and JMJD1A) was also attenuated . This methodological approach provides valuable insights into PIAS4's role in hypoxia signaling.

What methodologies are recommended for studying PIAS4's role in antiviral responses?

Recent research has revealed a novel role for PIAS4 as an intrinsic antiviral factor against herpes simplex virus 1 (HSV-1) infection . To investigate this function:

Subcellular localization during viral infection:

• Infect cells with HSV-1 at MOI of 0.1-10

• At different time points post-infection (1, 2, 4, 8 hours), fix and immunostain with anti-PIAS4 antibodies

• Co-stain with viral proteins (e.g., ICP0, ICP4) to track infection progression

• Use confocal microscopy to determine PIAS4's localization to viral genome entry sites or replication compartments

Functional studies:

• Deplete PIAS4 using validated siRNAs targeting exon 2 or exon 6

• Infect control and PIAS4-depleted cells with ICP0-null mutant HSV-1

• Quantify viral replication by plaque assays or qPCR of viral genes

• Compare with PML (promyelocytic leukemia protein) depletion to assess synergistic effects

Domain-specific recruitment analysis:

• Generate constructs expressing PIAS4 mutants lacking specific domains:

  • SAP domain (DNA binding)

  • PINIT domain (substrate recognition)

  • SP-RING domain (SUMO ligase activity)

  • SIM motifs (SUMO-interaction)

• Assess recruitment to viral genome entry sites and replication compartments using IF

• Use live-cell imaging with fluorescently tagged PIAS4 to track dynamics during infection

Research has shown that PIAS4 relocalizes to sites associated with viral DNA throughout infection, with SIM-dependent mechanisms at genome entry sites and SIM-independent mechanisms in replication compartments . These methodological approaches can reveal the mechanisms of PIAS4's antiviral activities.

How can researchers analyze PIAS4's E3 SUMO ligase activity in different experimental models?

PIAS4 functions as an E3 SUMO ligase, mediating the sumoylation of various substrate proteins. To analyze this enzymatic activity:

In vitro sumoylation assays:

• Express and purify recombinant PIAS4 protein

• Set up reaction mixtures containing:

  • E1 enzyme (SAE1/SAE2)

  • E2 enzyme (UBC9)

  • SUMO1, SUMO2, or SUMO3 proteins

  • ATP regeneration system

  • Purified substrate protein (e.g., GATA2, VHL, p53)

• Incubate at 30°C for 1-3 hours

• Analyze by Western blot using anti-SUMO and substrate-specific antibodies

Cellular sumoylation studies:

• Co-transfect cells with:

  • Tagged SUMO constructs (His6-SUMO or HA-SUMO)

  • PIAS4 expression vector or siRNA

  • Substrate protein

• Lyse cells in denaturing conditions (8M urea buffer)

• For His-tagged SUMO, perform Ni-NTA pulldown

• For HA-tagged SUMO, perform anti-HA immunoprecipitation

• Detect sumoylated substrates by Western blot

Domain mutant analysis:

• Generate PIAS4 mutants:

  • C342A (SP-RING domain mutant that abolishes E3 ligase activity)

  • Lys35 mutant (affects auto-sumoylation site)

• Compare wild-type and mutant PIAS4 in sumoylation assays

• Use CRISPR/Cas9 to generate endogenous PIAS4 domain mutants

In pancreatic cancer research, investigators have shown that PIAS4-mediated sumoylation of VHL leads to its oligomerization and reduced tumor suppressor activity against HIF1α . Similar methodological approaches can be applied to study PIAS4's E3 ligase activity toward other substrates in different biological contexts.

How can researchers address common technical challenges when using PIAS4 antibodies?

Researchers often encounter technical issues when working with PIAS4 antibodies. Here are methodological solutions to common problems:

Multiple bands in Western blot:

• PIAS4 can appear at 57-70 kDa due to post-translational modifications, particularly sumoylation

• Include N-ethylmaleimide (20 mM) in lysis buffers to preserve SUMO modifications

• Use gradient gels (4-15%) to better resolve different modified forms

• Validate bands using PIAS4 knockdown/knockout controls

• Consider that the 57 kDa band corresponds to the calculated molecular weight, while higher bands may represent modified forms

Weak or no signal in immunostaining:

• Optimize fixation (try 4% PFA and methanol fixation in parallel)

• Test different permeabilization methods (0.1-0.5% Triton X-100, 0.1% SDS, or methanol)

• Increase antibody concentration to 1:50 for initial optimization

• Use tyramide signal amplification for low-abundance detection

• Try heat-mediated antigen retrieval even for cell samples (citrate buffer pH 6.0)

High background in immunoprecipitation:

• Pre-clear lysates with protein A/G beads for 1 hour at 4°C

• Use more stringent wash buffers containing 250-500 mM NaCl

• Include 0.1% SDS in wash buffers to reduce non-specific binding

• Cross-link antibodies to beads to prevent heavy chain interference in blotting

• Use TrueBlot secondary antibodies to detect only native immunoglobulins

Inconsistent results between experiments:

• Standardize cell culture conditions, as PIAS4 expression can vary with cell density

• For hypoxia studies, tightly control O₂ levels and exposure times

• Maintain consistent protein loading (30-50 μg total protein per lane)

• Include positive control samples in each experiment (K-562 or HL-60 cells)

How should researchers interpret conflicting PIAS4 data in different cancer models?

Studies have reported seemingly contradictory roles for PIAS4 in different cancer types, particularly regarding its effects on hypoxia signaling. Here's how to methodically approach and interpret such conflicting data:

Methodological approach to conflicting data:

• Carefully analyze experimental conditions:

  • Cell type specificity: PIAS4 shows high expression in pancreatic cancer but may differ in other cancers

  • Oxygen tension: Compare normoxic vs. hypoxic conditions (1% vs. 21% O₂)

  • Time points: Acute vs. chronic responses may differ substantially

• Examine substrate specificity:

  • In pancreatic cancer, PIAS4 primarily targets VHL for sumoylation

  • In colon cancer, PIAS4 has been reported to directly sumoylate HIF1α

  • Use IP-mass spectrometry to identify predominant substrates in your model

• Consider context-dependent interactions:

  • Analyze co-expression of other PIAS family members that may compensate

  • Assess expression of desumoylating enzymes (SENPs) that might counteract PIAS4

  • Examine p53 status, as PIAS4-p53 interaction affects outcomes

• Validate with multiple approaches:

  • Use both gain-of-function (overexpression) and loss-of-function (siRNA) studies

  • Confirm findings with at least two different antibodies

  • Employ both in vitro and in vivo models when possible

Research has shown that in colon cancer, high PIAS4 expression was associated with enhanced HIF1α sumoylation and deactivation, while in pancreatic cancer, PIAS4 induction positively correlated with HIF1α activity through VHL suppression . These differences highlight the importance of cellular context in PIAS4 function.

What statistical approaches are appropriate for quantifying PIAS4 expression and localization changes?

Accurate quantification of PIAS4 requires appropriate statistical methods depending on the experimental technique:

Western blot quantification:

• Use densitometry software (ImageJ, Image Lab) to measure band intensity

• Normalize to appropriate loading controls (β-actin for whole cell, Lamin B for nuclear fractions)

• For comparing multiple conditions:

  • Perform one-way ANOVA with Tukey's post-hoc test for multiple comparisons

  • Use paired t-tests for before/after treatments on the same samples

• Present data as fold-change relative to control conditions

• For time-course experiments, use repeated measures ANOVA

Immunofluorescence quantification:

• For subcellular localization analysis:

  • Measure nuclear/cytoplasmic intensity ratio in at least 50-100 cells per condition

  • Use Pearson's correlation coefficient to quantify colocalization with other proteins

  • Apply Mann-Whitney U test for non-parametric data comparison

• For focal structures (e.g., PIAS4 at viral genome entry sites):

  • Count foci number per cell

  • Measure foci intensity and size

  • Use Poisson distribution-based statistics for count data

RT-qPCR expression analysis:

• Use 2^(-ΔΔCt) method with appropriate reference genes

• Employ multiple reference genes (GAPDH, β-actin, 18S rRNA)

• For pancreatic cancer studies, include at least 10-12 tumor samples and 6+ normal tissues

• Apply non-parametric tests (Wilcoxon signed-rank) for clinical sample comparisons

Tissue microarray analysis:

• Use H-score or Allred scoring systems for IHC quantification

• Employ blinded scoring by at least two independent pathologists

• Calculate inter-observer agreement using Cohen's kappa

• Correlate with patient data using Kaplan-Meier survival analysis and Cox regression

Research has shown that PIAS4 mRNA levels were elevated approximately three-fold in pancreatic cancer cell lines and six-fold in fresh pancreatic tumors compared to normal pancreas (P=0.0154) , demonstrating the importance of rigorous statistical analysis in PIAS4 research.

How can researchers investigate PIAS4's dual roles in innate and intrinsic immunity?

PIAS4 presents an intriguing research target due to its seemingly contradictory roles in immune responses. While PIAS proteins typically suppress innate immune signaling, PIAS4 has been identified as a positive regulator of intrinsic antiviral immunity . Investigating this duality requires sophisticated methodological approaches:

Comparative viral infection models:

• Challenge cells with different virus types:

  • DNA viruses (HSV-1, CMV, adenovirus)

  • RNA viruses (influenza, SARS-CoV-2)

  • Retroviruses (HIV)

• Monitor PIAS4 localization and activity using IF and biochemical approaches

• Generate time-course data to distinguish between early (intrinsic) and late (innate) immune responses

Pathway-specific dissection:

• Simultaneously monitor multiple immune pathways in PIAS4-depleted cells:

  • cGAS-STING (DNA sensing)

  • RIG-I/MDA5 (RNA sensing)

  • IFN receptor signaling

  • NF-κB activation

• Use reporter cell lines expressing luciferase under pathway-specific promoters

• Employ multiplexed cytokine profiling to assess downstream effects

Domain-specific mutant analysis:

• Create PIAS4 constructs with mutations in different functional domains:

  • SAP domain (DNA binding)

  • SIM motifs (SUMO interaction)

  • SP-RING domain (E3 ligase activity)

• Assess each mutant's ability to:

  • Restrict viral replication

  • Suppress or enhance IFN signaling

  • Localize to different subcellular compartments

• Identify domains responsible for pathway-specific functions

Research has demonstrated that PIAS4 is recruited to sites associated with HSV-1 genome entry through SIM-dependent mechanisms and accumulates in replication compartments through SIM-independent mechanisms . Understanding how the same protein can both enhance intrinsic immunity while suppressing innate immunity represents a significant research opportunity.

What methodologies can reveal PIAS4's role in DNA damage response and cancer therapy resistance?

PIAS4 functions in DNA damage response pathways by mediating sumoylation of repair proteins, potentially influencing cancer therapy resistance. To investigate this aspect:

DNA damage induction models:

• Treat cells with different DNA-damaging agents:

  • Ionizing radiation (Double-strand breaks)

  • Cisplatin (Interstrand crosslinks)

  • UV radiation (Pyrimidine dimers)

  • Hydroxyurea (Replication stress)

• Track PIAS4 recruitment to damage sites using live-cell imaging

• Quantify retention kinetics at damage sites in the presence/absence of key repair proteins

Repair pathway-specific analysis:

• Measure repair efficiency in PIAS4-depleted cells using:

  • Comet assay for global DNA damage

  • DR-GFP assay for homologous recombination

  • NHEJ reporter assay for non-homologous end joining

• Identify PIAS4-dependent sumoylation targets in each pathway using SUMO proteomics

• Create cellular systems with inducible expression of PIAS4 mutants during DNA damage

Therapeutic resistance studies:

• Generate isogenic cell lines with PIAS4 knockdown/overexpression

• Assess sensitivity to:

  • PARP inhibitors (olaparib)

  • Platinum compounds

  • Radiotherapy

  • Topoisomerase inhibitors

• Combine PIAS4 inhibition with established therapies to test for synergistic effects

PIAS4 has been implicated in the DNA damage response pathway and is thought to work in combination with PIAS1 for the productive association of repair factors like 53BP1, BRCA1, and RNF168 . Methodical investigation of these interactions may reveal PIAS4 as a potential therapeutic target for overcoming treatment resistance.

How can researchers address the emerging roles of PIAS4 in neurodegenerative diseases?

Recent findings suggest PIAS4 may play roles in neurodegenerative diseases through its regulation of protein homeostasis and stress responses . To investigate these emerging functions:

Protein aggregation models:

• Express neurodegenerative disease-associated proteins in cellular models:

  • Tau (Alzheimer's disease)

  • α-synuclein (Parkinson's disease)

  • Huntingtin (Huntington's disease)

• Manipulate PIAS4 levels using siRNA or overexpression

• Quantify aggregate formation using filter trap assays and microscopy

• Assess whether PIAS4-mediated sumoylation alters protein solubility and toxicity

Neuronal stress response studies:

• Subject primary neurons or differentiated iPSCs to:

  • Oxidative stress (H₂O₂, paraquat)

  • ER stress (tunicamycin, thapsigargin)

  • Proteasome inhibition (MG132)

• Monitor PIAS4 expression, localization, and activity changes

• Determine if PIAS4 modulation can protect against stress-induced neuronal death

In vivo neurodegeneration models:

• Generate conditional PIAS4 knockout mice using neuron-specific Cre lines

• Cross with established neurodegenerative disease models

• Assess:

  • Behavioral phenotypes

  • Histopathological markers

  • Protein aggregation burden

  • Neuroinflammatory signatures

The potential role of PIAS4 in neurodegenerative diseases represents an emerging research area. PIAS4 can interact with proteins like PARK7/DJ-1 involved in Parkinson's disease , and its SUMO E3 ligase activity may influence protein aggregation and clearance mechanisms central to neurodegeneration.