PARP11 Antibody

What is PARP11 and what are its known functions in cellular biology?

PARP11 (poly(ADP-ribose) polymerase family member 11), also known as ARTD11, is a mono-ADP-ribosyltransferase that mediates mono-ADP-ribosylation of target proteins . PARP11 functions in several key cellular processes:

• Regulates immune responses through the type I interferon pathway by modifying β-TrCP proteins

• Plays a crucial role in nuclear envelope stability and nuclear remodeling during spermiogenesis

• Acts as a key regulator of the immunosuppressive tumor microenvironment (TME)

• Participates in viral infection responses, though its effects vary by virus type

How do I select the appropriate PARP11 antibody for my specific research application?

Selection should be methodically approached based on:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, ELISA)

• Species reactivity: Confirm reactivity with your experimental model - common reactivity includes human and mouse, with some antibodies also recognizing rat and pig samples

• Epitope targeting: Consider antibodies targeting different regions if studying specific domains

• Validation data: Review published literature citing specific antibody clones

• Controls: Plan for appropriate positive controls (e.g., HeLa cells, PC-3 cells, mouse heart/spleen tissue)

What are the optimal conditions for detecting PARP11 in Western blot applications?

For robust and reproducible Western blot detection of PARP11:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors

  • Include phosphatase inhibitors if studying post-translational modifications

• Gel electrophoresis:

  • Load 20-50 μg total protein per lane

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

• Transfer parameters:

  • Semi-dry or wet transfer (wet preferred for larger proteins)

  • Transfer at 100V for 60-90 minutes in cold room

• Antibody incubation:

  • Block with 5% non-fat milk or BSA (if phospho-specific)

  • Primary antibody dilution: 1:500-1:2000

  • Incubate overnight at 4°C with gentle rocking

  • Expected molecular weight: ~30 kDa (observed) vs. calculated 39 kDa

• Detection considerations:

  • Enhanced chemiluminescence with longer exposure times may be needed

  • Consider using HeLa or PC-3 cells as positive controls

How should experimental protocols be modified for PARP11 detection in different cell types or tissues?

Protocol modifications should account for tissue-specific expression patterns and potential interfering factors:

• Cell line considerations:

  • Expression levels vary significantly between tissues/cell types

  • Highest expression observed in heart and spleen tissues

  • Cancer cells may show altered expression patterns

• Tissue-specific extraction methods:

  • Fibrous tissues: Consider specialized extraction buffers with higher detergent concentrations

  • Brain tissue: Use specialized extraction protocols to remove lipids

  • Fixed tissues: Optimize antigen retrieval (TE buffer pH 9.0 recommended for IHC)

• Background reduction strategies:

  • For high background in IHC: Extended blocking (2-3 hours) with serum from same species as secondary antibody

  • For non-specific bands in WB: Pre-adsorption with blocking peptides

• Signal enhancement approaches:

  • Tyramide signal amplification for low abundance detection

  • Biotin-streptavidin amplification systems for IHC

How does PARP11 function in tumor immunology and how can this be experimentally manipulated?

PARP11 emerges as a critical regulator of anti-tumor immunity with significant therapeutic implications:

• PARP11's role in tumor microenvironment:

  • Acts as a key regulator of immunosuppressive tumor microenvironment (TME)

  • Induced in intratumoral cytotoxic T lymphocytes (CTLs) by TME factors

  • Catalyzes the mono-ADP-ribosylation of β-TrCP proteins

  • Promotes downregulation of type I interferon receptor IFNAR1

  • Associated with T cell exhaustion in tumor models

• Experimental manipulation approaches:

  • Genetic: CRISPR/Cas9 knockout of PARP11 in T cells

  • Pharmacological: PARP11 inhibitors like ITK7 (IC50 = 14 nM)

  • CAR-T engineering: Fourth-generation CARs with shRNA against PARP11

• Experimental readouts:

  • IFNAR1 surface levels by flow cytometry

  • β-TrCP levels and ADP-ribosylation by Western blot

  • T cell cytotoxicity assays against tumor cells

  • In vivo tumor growth in PARP11 knockout models

What methodologies effectively assess PARP11's role in viral infection models?

PARP11 demonstrates virus-specific effects that require careful experimental design:

• Cell culture infection models:

  • PARP11 inhibition/knockout promotes PRV (pseudorabies virus) infection

  • PARP11 knockout inhibits VSV and Sendai virus replication

  • PARP11 suppresses Zika virus replication in cooperation with PARP12

• Recommended experimental approaches:

  • Generate PARP11 knockout cell lines using CRISPR/Cas9

  • Pharmacological inhibition with ITK7

  • siRNA knockdown of PARP11

  • Measure viral titers using TCID50 assays

  • Quantify viral genome copies by qPCR

  • RNA-seq analysis to identify affected pathways

• Mechanistic investigation methods:

  • Analysis of mRNA export pathways (NXF1, CRM1)

  • Examination of autophagy markers during infection

  • Assessment of mTOR pathway activation

  • IFN-I-induced STAT1 activation analysis

How can researchers effectively study PARP11-mediated post-translational modifications and their functional consequences?

Investigating PARP11's catalytic activity requires specialized techniques:

• ADP-ribosylation detection methods:

  • In vitro ADP-ribosylation assays using recombinant proteins

  • Immunoprecipitation followed by detection with anti-PAR antibodies

  • Mass spectrometry to identify specific ADP-ribosylation sites

  • Western blot analysis for mobility shifts in target proteins

• Target identification approaches:

  • Proteomic analysis comparing WT vs. PARP11-knockout cells

  • Proximity labeling techniques (BioID, APEX)

  • Candidate approach focusing on β-TrCP and other reported targets

• Functional consequence assessment:

  • β-TrCP stability and availability to ubiquitinate IFNAR1

  • IFNAR1 degradation and IFN1 signaling pathway activity

  • Downstream effects on T cell function and anti-tumor activity

What are the most common technical challenges when studying PARP11 and how can they be addressed?

Researchers frequently encounter specific challenges when investigating PARP11:

• Antibody specificity issues:

  • Problem: Cross-reactivity with other PARP family members

  • Solution: Validate specificity using PARP11 knockout samples as negative controls

  • Solution: Use multiple antibodies targeting different epitopes

• Variable expression levels:

  • Problem: PARP11 expression can be induced by stimuli like adenosine and PGE2

  • Solution: Carefully control experimental conditions

  • Solution: Include time-course analyses after stimulation

• Functional redundancy with other PARPs:

  • Problem: Compensatory mechanisms in knockout models

  • Solution: Consider double knockout approaches

  • Solution: Use pharmacological inhibitors with appropriate controls

• Quantifying subtle phenotypes:

  • Problem: Effects may be context-dependent or partial

  • Solution: Use sensitive readouts (e.g., reporter assays)

  • Solution: Perform experiments in relevant physiological contexts (e.g., tumor microenvironment)

How can researchers design experiments to investigate PARP11's role in CAR-T cell therapy enhancement?

Based on emerging evidence, researchers can systematically explore PARP11 manipulation in CAR-T cells:

• Experimental design considerations:

  • Generate CAR-T cells from PARP11-knockout or PARP11-inhibited T cells

  • Engineer CAR constructs expressing shRNA against PARP11

  • Combine pharmacological PARP11 inhibition with CAR-T therapy

  • Include appropriate controls (e.g., PARP11-null IFNAR1-null double knockouts)

• Functional assessment methods:

  • In vitro cytotoxicity assays against target cells in presence of TME factors

  • Flow cytometric analysis of exhaustion markers (TIM3, PD-1, LAG-3)

  • CAR-T cell persistence measurement in blood and tumors

  • Tumor growth inhibition in xenograft models

• Mechanism investigation approaches:

  • IFNAR1 surface levels and stability measurement

  • β-TrCP levels and ADP-ribosylation status

  • Transcriptomic analysis of IFN-stimulated genes

  • Combinatorial approaches with immune checkpoint blockade