PRMT9 Antibody, FITC conjugated

Basic Research Questions

• What is PRMT9 and what are its primary biological functions?

PRMT9 is a type II protein arginine methyltransferase that catalyzes the formation of symmetric dimethylarginine (SDMA) on target proteins. It contains two S-adenosylmethionine binding motifs and three tetratricopeptide repeats (TPRs) . The primary known function of PRMT9 is methylation of the splicing factor SF3B2/SAP145 at arginine 508, which regulates alternative splicing of pre-mRNA . Recent research has expanded our understanding of PRMT9's roles to include:

• Modulation of the splicing factor SAP145/SAP149 complex

• Attenuation of MAVS signaling in antiviral immunity

• Cancer cell survival and proliferation in acute myeloid leukemia (AML)

• Regulation of neuronal development and synapse formation

PRMT9 is primarily localized in the cytoplasm, as confirmed by subcellular localization studies using monoclonal antibodies .

• How do FITC-conjugated PRMT9 antibodies differ from unconjugated versions in research applications?

FITC-conjugated PRMT9 antibodies offer several distinct advantages over unconjugated versions:

• Direct detection without secondary antibodies, reducing background and cross-reactivity issues

• Single-step staining procedures for immunofluorescence and flow cytometry

• Compatibility with live-cell imaging approaches

• Ability to perform multiplexed staining with other fluorophore-conjugated antibodies

• Quantitative analysis through fluorescence intensity measurements

Methodological considerations when using FITC-conjugated versus unconjugated antibodies:

When designing experiments, researchers should consider that FITC has excitation/emission maxima around 494/518 nm and protect the conjugate from prolonged light exposure.

• What are the optimal fixation and permeabilization methods for PRMT9 immunofluorescence staining?

For optimal immunofluorescence staining of PRMT9:

Fixation methods (in order of preference):

• 4% paraformaldehyde (15 minutes at room temperature) - preserves both protein localization and cellular morphology

• Ice-cold methanol (10 minutes at -20°C) - better for preserving cytoskeletal components but may affect epitope recognition

• Acetone (10 minutes at -20°C) - rapid fixation but may distort some cellular structures

Permeabilization options:

• 0.1% Triton X-100 in PBS (10 minutes at room temperature)

• 0.1% saponin in PBS (20 minutes at room temperature) - gentler and may better preserve membrane structures

• 0.5% Tween-20 in PBS (15 minutes at room temperature)

Because PRMT9 has been primarily detected in the cytoplasm with some specific interactions in nuclear speckles , researchers should optimize permeabilization conditions to ensure access to both compartments. Always include controls with varying fixation/permeabilization conditions to determine optimal protocols for your specific cell type and antibody.

• What are the primary applications for FITC-conjugated PRMT9 antibodies?

FITC-conjugated PRMT9 antibodies are versatile tools across multiple research applications:

• Immunofluorescence microscopy: Visualize PRMT9 distribution in fixed cells and tissues, with particular utility for co-localization studies with splicing factors like SF3B2/SAP145

• Flow cytometry: Quantitatively assess PRMT9 expression levels across heterogeneous cell populations, particularly valuable in cancer research where PRMT9 levels are elevated in leukemia stem cells

• Fluorescence-activated cell sorting (FACS): Isolate cell populations based on PRMT9 expression levels for downstream functional analyses

• Immunoprecipitation verification: Confirm successful immunoprecipitation of PRMT9 complexes when performing protein interaction studies

• In situ proximity ligation assays: Investigate protein-protein interactions between PRMT9 and potential binding partners

Optimal dilutions for applications (based on comparable antibodies):

• Immunofluorescence: 1:100-1:500

• Flow cytometry: 1:50-1:200

• Western blot validation: 1:1000-1:2000

Advanced Research Questions

• How can researchers design experiments to investigate PRMT9's role in cancer progression using FITC-conjugated antibodies?

Based on recent findings implicating PRMT9 in cancer survival mechanisms, particularly in AML , researchers can design experiments using FITC-conjugated PRMT9 antibodies through several methodological approaches:

A. Flow cytometry-based experimental design:

• Use FITC-conjugated PRMT9 antibodies to quantify expression across primary patient samples and cancer cell lines

• Sort cells based on PRMT9 expression levels (high vs. low) for functional assays

• Implement a multiparameter analysis combining PRMT9-FITC with markers for:

  • Cell cycle (propidium iodide or DAPI)

  • Apoptosis (Annexin V)

  • Cancer stem cell markers (CD34+CD38- for AML)

  • DNA damage (γH2AX)

B. High-content imaging approach:

• Establish cell models with modulated PRMT9 expression (knockdown, knockout, overexpression)

• Stain with FITC-conjugated PRMT9 antibodies alongside markers for:

  • Type I interferon response factors

  • DNA damage sensors (cGAS/STING pathway components)

  • Immune checkpoint proteins (PD-1/PD-L1)

• Quantify correlations between PRMT9 levels and these markers

C. Therapeutic response monitoring:

• Treat cells with PRMT9 inhibitors at varying concentrations

• Use FITC-conjugated PRMT9 antibodies to monitor changes in:

  • Expression levels

  • Subcellular localization

  • Association with binding partners

• Combine with functional readouts (cell survival, DNA damage, immune activation)

Recent research has shown that "PRMT9 inhibition promoted DNA damage and activated cyclic GMP-AMP synthase, which underlies the type I IFN response" , suggesting experimental designs should incorporate DNA damage markers and immune response readouts.

• What methodological approaches can be used to study PRMT9-mediated methylation of splicing factors?

Investigating PRMT9-mediated methylation of splicing factors requires specialized methodological approaches:

A. In vitro methylation assays:

• Express recombinant PRMT9 and substrate (e.g., SF3B2 fragment)

• Perform methylation reactions using:

  • Tritiated S-adenosylmethionine ([³H]-SAM) for radiometric detection

  • Non-radioactive SAM with methyl-specific antibody detection

• Validate using recombinant SF3B2 with site-directed mutations at Arg508

This approach can be implemented using the PRMT9 Homogeneous Assay Kit format described in the AlphaLISA® format , which includes:

• Purified recombinant PRMT9

• S-adenosylmethionine

• Primary antibody

• SAP145 (amino acids 401-550)

B. Mass spectrometry-based detection:

• Immunoprecipitate PRMT9 and its binding partners

• Digest and perform LC-MS/MS analysis

• Search for methylation modifications at specific arginine residues

• Compare methylation patterns in control vs. PRMT9-depleted samples

C. Site-specific mutation approach:
Implement the mutation strategy described in Yang et al. , where each arginine residue in the SF3B2 F3 fragment was replaced with lysine. This revealed R508 as the specific methylation target, as "when R508 was mutated to lysine, methylation was greatly diminished, indicating the specificity of PRMT9 for this residue" .

D. Alternative splicing functional readouts:

• Establish PRMT9 knockdown or knockout systems

• Analyze splicing patterns using RNA-seq

• Focus on genes with documented alternative splicing changes, such as the ~1900 genes identified in hippocampal neurons of PRMT9 conditional knockout mice

• Validate with minigene reporter constructs

• How can FITC-conjugated PRMT9 antibodies be used to investigate the relationship between PRMT9 and immune response pathways?

Recent research has identified PRMT9 as a negative regulator of innate antiviral immunity , providing a foundation for experimental approaches using FITC-conjugated PRMT9 antibodies:

A. Viral infection experimental system:

• Establish infection models using RNA viruses (SeV, VSV) as demonstrated in previous research

• Track PRMT9 expression and localization using FITC-conjugated antibodies during infection time course

• Co-stain with markers for:

  • MAVS aggregation (which PRMT9 inhibits through arginine methylation)

  • Type I interferon signaling components

  • Viral proteins

B. MAVS methylation analysis:

• Generate cells with wildtype PRMT9, PRMT9 knockout, or enzymatically inactive PRMT9 (using the L182A/D183A/I184A/G185A quadruple mutations described in previous research)

• Use FITC-conjugated PRMT9 antibodies to confirm expression levels

• Quantify PRMT9-MAVS co-localization under different stimulation conditions

• Correlate with MAVS arginine methylation at Arg41 and Arg43

C. Flow cytometry immune profiling:

• Isolate immune cells from control and PRMT9 conditional knockout mice

• Implement a multi-parameter staining panel including:

  • FITC-conjugated PRMT9 antibody

  • Markers for immune cell subsets

  • Activation markers

  • Intracellular cytokine staining for IFN-α/β

• Analyze correlation between PRMT9 expression and immune activation status

Data from myeloid-specific PRMT9 knockout mice indicates that "infection of Prmt9 CKO peritoneal macrophages with SeV or stimulation with 5'PPP RNA led to a significant increase in the expression of Ifnb1, Ifnα4, and Cxcl10 mRNA" , providing a foundation for investigating this regulatory axis.

• What are the key technical considerations for validating the specificity of FITC-conjugated PRMT9 antibodies?

Thorough validation of FITC-conjugated PRMT9 antibodies is essential for reliable research outcomes:

A. Genetic approach validation:

• Test antibody on samples from:

  • PRMT9 knockout models (complete or conditional)

  • PRMT9 knockdown cells (siRNA or shRNA)

  • PRMT9 overexpression systems

• Confirm specific signal reduction/increase corresponding to genetic manipulation

B. Biochemical validation protocols:

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide (such as "HTKSLDIEIP KHIPERVSLV VTETVDAGLF GEGIVESLIH AWEHLLLQPK TKGESANCEK YGKVIPASAV IFGMAVECAE IRRHHRVGIK DIAGIHLPT" )

  • Compare staining with and without peptide competition

• Cross-reactivity assessment:

  • Test against other PRMT family members, particularly PRMT5 (the other type II PRMT)

  • Use Western blot to confirm specific recognition of ~94-105 kDa protein band

C. Application-specific controls:

• For immunofluorescence:

  • Include isotype control antibody at equivalent concentration

  • Compare staining patterns against published subcellular localization (primarily cytoplasmic)

  • Perform fluorescence minus one (FMO) controls

• For flow cytometry:

  • Implement titration series to determine optimal antibody concentration

  • Include unstained, isotype, and secondary-only controls

  • Validate with cells known to express different levels of PRMT9

D. Advanced validation approaches:

• Correlate FITC signal with protein detection by other methods (Western blot, mass spectrometry)

• Confirm co-localization with known PRMT9 interacting partners (e.g., SF3B2/SAP145)

• Verify recognition of recombinant PRMT9 with different tags (as used in experimental systems)

• How can researchers design experiments to investigate PRMT9's role in neuronal development using FITC-conjugated antibodies?

Recent research has uncovered PRMT9's crucial role in neuronal development and synaptic function , providing a framework for experimental approaches:

A. Primary neuron culture analysis:

• Establish primary hippocampal or cortical neurons from control and conditional PRMT9 knockout mice

• Use FITC-conjugated PRMT9 antibodies for:

  • Expression time course during neuronal development

  • Subcellular localization studies

  • Co-localization with synaptic markers

• Quantitative analysis of:

  • Dendritic spine density and morphology

  • Synaptic protein clustering

  • Correlation with electrophysiological parameters

B. Brain tissue immunohistochemistry protocol:

• Prepare brain sections from different developmental stages

• Implement dual immunofluorescence with:

  • FITC-conjugated PRMT9 antibody

  • Markers for excitatory/inhibitory synapses

  • Neuronal subtypes and glial cells

• Quantitative analysis across brain regions and developmental stages

C. Functional correlation experimental design:

• Generate neurons with PRMT9 knockdown or expression of the G189R mutation (which "abolishes PRMT9 methyltransferase activity and reduces its protein stability")

• Use FITC-conjugated PRMT9 antibodies to confirm expression/localization

• Correlate with:

  • Alternative splicing patterns of the ~1900 genes affected by PRMT9 knockout

  • Electrophysiological recordings of synaptic function

  • Behavioral assays in animal models

D. SF3B2 methylation-dependency analysis:

• Generate neurons expressing wildtype SF3B2 or methylation-deficient R508K mutant

• Use FITC-conjugated PRMT9 antibodies to track enzyme localization

• Analyze impact on:

  • The "methylation-sensitive protein–RNA interaction between the arginine 508 (R508) of SF3B2 and the pre-mRNA anchoring site"

  • Downstream splicing patterns

  • Neuronal morphology and function

• What experimental approaches can be used to study the therapeutic potential of targeting PRMT9 in cancer?

Building on the discovery that "targeting PRMT9-mediated arginine methylation suppresses cancer immunity evasion" , researchers can implement several experimental strategies:

A. Inhibitor screening and validation protocol:

• Test compounds like those described by Carosso et al. with IC₅₀ values against PRMT9

• Use FITC-conjugated PRMT9 antibodies to:

  • Confirm target engagement in living cells

  • Track changes in PRMT9 localization after inhibitor treatment

  • Monitor potential protein degradation

• Establish dose-response relationships across different cancer cell types

B. Combination therapy experimental design:

• Based on the finding of "synergy of a PRMT9 inhibitor with anti-programmed cell death protein 1 in eradicating AML" :

  • Treat cells with PRMT9 inhibitors alone or in combination with immune checkpoint inhibitors

  • Use FITC-conjugated PRMT9 antibodies to monitor target expression

  • Analyze immune activation markers

• Implement in vitro co-culture systems with cancer cells and immune effectors

C. Mechanistic analysis of PRMT9 inhibition:

• Establish assays to monitor:

  • DNA damage response (following the finding that "PRMT9 inhibition promoted DNA damage")

  • cGAS-STING pathway activation ("activation of cyclic GMP-AMP synthase, which underlies the type I IFN response")

  • Type I interferon signaling

• Use FITC-conjugated PRMT9 antibodies for sorting cells with varying PRMT9 expression

• Correlate PRMT9 levels with response to therapy

D. In vivo experimental approach:

• Establish xenograft or syngeneic tumor models

• Treat with PRMT9 inhibitors alone or in combination with immunotherapy

• Use FITC-conjugated PRMT9 antibodies for:

  • Flow cytometric analysis of tumor cells

  • Immunofluorescence microscopy of tissue sections

  • Monitoring changes in PRMT9 expression during treatment

This comprehensive experimental approach builds on the finding that "PRMT9 functions in survival and immune evasion of both LSCs and non-LSCs; targeting PRMT9 may represent a potential anticancer strategy" .

Technical Notes

For consistent results with FITC-conjugated PRMT9 antibodies, researchers should:

• Store antibodies at -20°C, protected from light

• Avoid repeated freeze-thaw cycles

• Optimize antibody concentration for each specific application and cell type

• Include appropriate controls in every experiment

• Be aware that FITC fluorescence is sensitive to pH changes