PPM1G Antibody

What is PPM1G and why is it important to study?

PPM1G, also known as Protein Phosphatase 2C isoform gamma (PP2C-gamma), is a member of the PP2C family of Ser/Thr phosphatases. It functions primarily as a magnesium/manganese-dependent enzyme that dephosphorylates target proteins, thereby regulating their activity in various cellular processes. PPM1G plays crucial roles in cell cycle regulation, stress responses, and immune signaling pathways .

The protein has several synonyms including PP2CG, PPP2CG, and PP2CGAMMA, and while its calculated molecular weight is 59kDa, it is often observed at 70-75kDa in experimental conditions due to post-translational modifications. PPM1G is primarily localized in the cytoplasm and can also be found associated with membranes through lipid anchors .

Recent research has revealed that PPM1G functions as a negative regulator of innate immune signaling pathways, particularly those mediated by cytosolic DNA and RNA sensing mechanisms. This regulatory function helps balance the intensity of antiviral responses, making PPM1G an important target for immunological and viral research .

What types of PPM1G antibodies are available for research use?

Several types of PPM1G antibodies are available for research, each with different characteristics and applications:

These antibodies vary in their specific epitope recognition, which may impact their performance in different applications. When selecting a PPM1G antibody, researchers should consider the specific epitope targeted, the validation data available, and the intended experimental application .

What applications are PPM1G antibodies validated for?

PPM1G antibodies have been validated for multiple research applications with specific performance characteristics:

The specific validation data for each antibody should be reviewed before use. For example, the HPA035531 antibody from Sigma-Aldrich has undergone enhanced validation through independent and orthogonal RNAseq validation methods, providing additional confidence in its specificity .

What species reactivity do PPM1G antibodies typically have?

PPM1G antibodies demonstrate cross-reactivity with samples from multiple species, which is valuable for comparative studies between human and animal models:

This cross-species reactivity reflects the high degree of conservation of PPM1G across mammalian species. When using these antibodies in non-human models, it's important to verify the expected molecular weight and staining patterns, as slight variations may occur between species .

How does PPM1G regulate innate immune signaling pathways?

PPM1G functions as a negative regulator of innate immune pathways by targeting key components of both cytosolic DNA and RNA sensing mechanisms. According to recent research published in Science Advances, PPM1G plays a critical role in balancing the intensity of antiviral responses through multiple mechanisms .

Mechanistically, PPM1G acts by:

• Dephosphorylating activated (phosphorylated) STING and MAVS, key adaptor proteins in the DNA and RNA sensing pathways respectively

• Reducing the downstream phosphorylation and dimerization of IRF3, a critical transcription factor for interferon production

• Consequently suppressing the expression of type I interferons (IFNβ) and interferon-stimulated genes (ISGs)

Experimental evidence from the study shows that overexpression of PPM1G significantly inhibits IFNβ promoter activity and mRNA expression. Additionally, PPM1G markedly decreases IRF3 phosphorylation and dimerization when cells are stimulated with poly(dA:dT) (DNA mimetic) or poly(I:C) (RNA mimetic) .

Conversely, knockdown of PPM1G enhances IFNβ production in response to poly(dA:dT), poly(I:C), HSV-1 (DNA virus), or Sendai virus (RNA virus). PPM1G knockout cells show significantly higher IFNβ and ISG56 mRNA levels in response to stimuli, compared to wild-type cells, further confirming its regulatory role .

How can PPM1G antibodies be used to study viral immune evasion mechanisms?

PPM1G antibodies are valuable tools for investigating viral immune evasion strategies, particularly following the discovery that certain viruses exploit host PPM1G to suppress immune responses. The Science Advances study reveals that Kaposi's sarcoma-associated herpesvirus (KSHV) hijacks the host PPM1G system for immune evasion .

Researchers can use PPM1G antibodies in several methodological approaches to study such mechanisms:

• Co-immunoprecipitation (Co-IP) experiments:

  • Use PPM1G antibodies (e.g., ab70794 at 3 μg/mg lysate) to pull down protein complexes

  • Analyze interactions between PPM1G, viral proteins (e.g., KSHV ORF33), and immune signaling components (STING, MAVS)

  • Compare protein complexes in infected versus uninfected cells

• Western blotting analysis:

  • Use PPM1G antibodies to detect changes in PPM1G expression during viral infection

  • Monitor the phosphorylation status of STING, MAVS, and IRF3 in the presence/absence of viral proteins

  • Compare results across different viral infection models (DNA viruses like HSV-1 vs. RNA viruses like Sendai virus)

• Immunofluorescence microscopy:

  • Use PPM1G antibodies for immunofluorescence (e.g., HPA035531 at 0.25-2 μg/mL)

  • Visualize the co-localization of PPM1G with viral proteins and immune signaling components

  • Track the translocation of PPM1G during different stages of viral infection

• Functional assays combining antibody validation:

  • Correlate PPM1G antibody signals with functional readouts (IFNβ production, viral replication)

  • Validate the specificity of observed effects through PPM1G knockdown/knockout controls

  • Compare results across multiple viral systems to identify common evasion mechanisms

These approaches can reveal how different viruses may exploit PPM1G to evade host immunity and could potentially identify novel therapeutic targets for antiviral development .

What are the key phosphorylation targets of PPM1G in immune signaling?

The Science Advances study identifies several key phosphorylation targets of PPM1G in immune signaling pathways that are critical for understanding its regulatory function:

• STING (Stimulator of Interferon Genes):

  • PPM1G dephosphorylates phosphorylated STING (p-STING)

  • STING is a crucial adaptor protein in the cytosolic DNA sensing pathway

  • Dephosphorylation by PPM1G suppresses STING-dependent signaling

• MAVS (Mitochondrial Antiviral Signaling protein):

  • PPM1G dephosphorylates phosphorylated MAVS (p-MAVS)

  • MAVS is a key adaptor protein in the cytosolic RNA sensing pathway

  • This dephosphorylation limits MAVS-mediated antiviral signaling

• Indirect regulation of IRF3 (Interferon Regulatory Factor 3):

  • While not necessarily a direct substrate, PPM1G activity leads to:

  • Reduced IRF3 phosphorylation

  • Decreased IRF3 dimerization

  • Suppressed IRF3-dependent gene expression

Experimental evidence shows that PPM1G knockout enhances responses to both DNA stimuli (poly(dA:dT)) and RNA stimuli (poly(I:C), SeV), with markedly increased phosphorylation of IRF3 in PPM1G-deficient cells. These findings establish PPM1G as a critical regulator targeting multiple components of innate immune signaling pathways .

How does the molecular weight of PPM1G observed in experiments differ from the predicted weight?

An interesting discrepancy researchers should be aware of is the difference between the calculated molecular weight of PPM1G and what is typically observed in experimental conditions:

This discrepancy between predicted and observed molecular weights could be attributed to several factors:

• Post-translational modifications:

  • Phosphorylation, glycosylation, or other modifications can significantly alter protein migration in SDS-PAGE

  • PPM1G has multiple potential modification sites that may be differentially regulated

• Protein isoforms:

  • Alternative splicing may generate different isoforms with varying molecular weights

  • Different cell types or conditions might express different isoforms

• Protein complexes:

  • Incomplete denaturation may result in PPM1G remaining in partial complexes

  • The 130 kDa band observed with ab70794 could represent such a complex or dimer

When designing experiments and analyzing results, researchers should anticipate these variations in PPM1G molecular weight and consider including appropriate controls to confirm antibody specificity .

What are the optimal conditions for using PPM1G antibodies in Western blotting?

Successful Western blotting with PPM1G antibodies requires optimization of several parameters. Based on the available data, here are detailed recommendations:

• Sample preparation:

  • Use whole cell lysates (HeLa, 293T cells work well as positive controls)

  • Include protease and phosphatase inhibitors in lysis buffers

  • Load 5-50 μg of protein lysate per lane (optimization may be required)

• Antibody selection and dilution:

  • ab70794: Use at 0.04 μg/mL concentration

  • CAB20959: Use at 1:100 - 1:500 dilution

  • HPA035531: Use at 0.04-0.4 μg/mL concentration

• Gel and transfer conditions:

  • Use 8-10% gels for optimal resolution around the 59-75 kDa range

  • Transfer proteins to PVDF or nitrocellulose membranes (PVDF may provide better results for phosphatases)

• Detection considerations:

  • ECL (Enhanced Chemiluminescence) works well with these antibodies

  • Exposure times as short as 10 seconds have been shown to be sufficient with ab70794

  • Expect bands at 70-75 kDa rather than the calculated 59 kDa

  • Some antibodies may detect additional bands at higher molecular weights (e.g., 130 kDa with ab70794)

• Controls to include:

  • Positive control: 293T cell lysate works well for most PPM1G antibodies

  • Loading control: Standard housekeeping proteins like GAPDH or β-actin

  • Negative control: Consider PPM1G knockdown or knockout samples where available

These conditions should be further optimized based on the specific experimental system, cell types, and antibody being used .

How can the specificity of PPM1G antibodies be validated?

Validating the specificity of PPM1G antibodies is crucial for ensuring reliable research results. Here are comprehensive methods for validating PPM1G antibodies:

• Genetic validation approaches:

  • Test in PPM1G knockout or knockdown cells

  • The signal should be significantly reduced or absent in these systems

  • The Science Advances study utilized PPM1G knockout cells that could serve as valuable controls

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • For ab70794, use the synthetic peptide from human PPM1G

  • For CAB20959, use recombinant fusion protein (amino acids 301-450)

  • The specific signal should be blocked or reduced

• Orthogonal validation:

  • Compare protein expression with mRNA expression data

  • Sigma-Aldrich's HPA035531 has undergone orthogonal RNAseq validation

  • Correlate antibody signal with mRNA levels across different cell types

• Multiple antibody approach:

  • Use different antibodies targeting distinct epitopes of PPM1G

  • Compare results between polyclonal (ab70794, HPA035531) and monoclonal (CAB20959) antibodies

  • Similar patterns across different antibodies increase confidence in specificity

• Functional correlation:

  • Link antibody signal to known PPM1G functions

  • For example, correlate PPM1G expression with IFNβ and ISG production as shown in the Science Advances study

  • Experimental interventions that alter PPM1G function should result in predictable changes in antibody signal

These validation approaches should be combined to provide comprehensive evidence of antibody specificity before proceeding with advanced applications .

What are the considerations for using PPM1G antibodies in immunoprecipitation experiments?

Immunoprecipitation (IP) is a valuable technique for studying protein-protein interactions and post-translational modifications of PPM1G. Based on the search results, here are detailed recommendations for optimizing PPM1G antibodies in IP experiments:

• Antibody selection:

  • ab70794 from Abcam has been validated for IP

  • The recommended amount is 3 μg of antibody per mg of cell lysate

• Lysate preparation:

  • Use appropriate lysis buffers that maintain protein-protein interactions

  • Include protease inhibitors and phosphatase inhibitors (especially important when studying phosphorylation events)

  • Typical lysate concentrations: 1 mg of total protein per IP reaction

• IP protocol optimization:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate cleared lysate with PPM1G antibody (3 μg/mg lysate) overnight at 4°C

  • For rabbit antibodies like ab70794, use Protein A or Protein A/G beads

  • Perform 4-5 washes with lysis buffer or TBS containing 0.1% detergent

• Controls and validation:

  • Include IgG control: Use matching concentration of non-specific IgG

  • Include input control: Load 5-10% of pre-IP lysate

  • For detecting PPM1G after IP, load approximately 20% of IP sample per lane for Western blot

  • Use ECL detection with exposure times as short as 1 second

• Application-specific considerations:

  • For studying PPM1G-STING/MAVS interactions :

    • Consider crosslinking to preserve transient interactions

    • Use gentler lysis conditions to maintain protein complexes

    • Include both infected and uninfected samples for comparison

• Troubleshooting:

  • If background is high: Increase washing stringency or pre-clearing time

  • If signal is low: Increase antibody or lysate amount, reduce wash stringency

  • If non-specific bands appear: Use more specific elution methods or increase antibody specificity

These optimized conditions should facilitate successful IP experiments with PPM1G antibodies for studying its interactions and modifications .

What are the considerations for using PPM1G antibodies in immunohistochemistry?

Using PPM1G antibodies in immunohistochemistry (IHC) requires careful optimization to ensure specific staining and reliable results:

• Antibody selection:

  • Both ab70794 from Abcam and HPA035531 from Sigma-Aldrich are validated for IHC-P (paraffin-embedded tissues)

  • HPA035531 has been extensively tested on 44 normal human tissues and 20 common cancer types as part of the Human Protein Atlas project

• Recommended dilutions:

  • For HPA035531, the recommended dilution range is 1:1000-1:2500

  • Titration experiments should be performed to determine optimal dilution for specific tissue types

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) with citrate buffer or EDTA buffer is typically effective

  • The optimal pH and buffer composition may need to be determined empirically

• Detection systems:

  • DAB (3,3′-Diaminobenzidine) is commonly used as a chromogen

  • For fluorescent detection, appropriate secondary antibodies should be selected based on the host species (rabbit for all PPM1G antibodies discussed)

• Controls:

  • Positive controls: Include tissues known to express PPM1G (e.g., HeLa cells, 293T cells)

  • Negative controls: Include serial sections with primary antibody omitted

  • Consider using PPM1G knockout or knockdown samples as additional specificity controls

• Interpretation:

  • Expect primarily cytoplasmic staining based on PPM1G's reported localization

  • Also consider membrane association due to lipid anchoring

  • Evaluate staining pattern, intensity, and distribution across different cell types

• Troubleshooting:

  • High background: Try longer blocking steps or different blocking agents

  • Weak signal: Optimize antigen retrieval, increase antibody concentration, or use signal amplification

  • Non-specific staining: Increase antibody dilution or use more stringent washing

These considerations should help researchers optimize IHC protocols for reliable detection of PPM1G in tissue sections .

How can PPM1G antibodies be used to study viral infection mechanisms?

PPM1G antibodies are valuable tools for studying viral infection mechanisms, particularly focusing on immune evasion strategies. Based on the Science Advances study, here's a methodological approach for applying PPM1G antibodies in viral research:

• Investigating viral manipulation of innate immunity:

  • Use PPM1G antibodies to detect changes in PPM1G expression during viral infection via Western blotting

  • Track PPM1G recruitment to specific cellular compartments during infection using immunofluorescence

  • Compare PPM1G-dependent signaling in cells infected with different viruses

• Studying virus-specific mechanisms:

  • For KSHV (as revealed in the Science Advances study):

    • Use PPM1G antibodies to detect interactions between viral ORF33 protein and host PPM1G

    • Monitor PPM1G recruitment to STING/MAVS complexes in the presence of viral proteins

    • Compare these mechanisms across different herpesviruses

• Methodological approach to viral immune evasion:

  • Co-immunoprecipitation experiments:

    • Use PPM1G antibodies to pull down protein complexes from virus-infected cells

    • Analyze viral proteins that co-precipitate with PPM1G

    • Identify host factors in the PPM1G-viral protein complex

  • Phosphorylation studies:

    • Use phospho-specific antibodies alongside PPM1G antibodies

    • Monitor the phosphorylation status of STING, MAVS, and IRF3 in infected cells

    • Correlate with PPM1G activity and localization

• Functional studies:

  • PPM1G knockdown/knockout experiments:

    • Deplete PPM1G using siRNA or CRISPR-Cas9

    • Measure the impact on viral replication

    • Quantify changes in interferon production and ISG expression

    • Research indicates that inhibition of PPM1G expression improves antiviral responses against both DNA and RNA viruses

The Science Advances study demonstrates that PPM1G restricts both cytosolic DNA and RNA sensing pathways, which viruses exploit for immune evasion. Using PPM1G antibodies to dissect these mechanisms can provide valuable insights into viral pathogenesis and potential therapeutic targets .

What role does PPM1G play in regulating the STING and MAVS signaling pathways?

PPM1G plays a critical regulatory role in both STING and MAVS-mediated signaling pathways, which are central to innate immune responses against pathogens. According to the Science Advances study, PPM1G functions as a negative regulator of these pathways through the following mechanisms :

STING-mediated signaling (DNA sensing pathway):

• STING becomes phosphorylated upon activation by cytosolic DNA or cyclic dinucleotides

• PPM1G counteracts this activation by dephosphorylating phosphorylated STING (p-STING)

• This dephosphorylation dampens STING-dependent signaling, reducing type I interferon production

• KSHV tegument protein ORF33 enhances this process by recruiting PPM1G to STING

MAVS-mediated signaling (RNA sensing pathway):

• MAVS becomes phosphorylated following recognition of viral RNA by RIG-I-like receptors

• PPM1G dephosphorylates phosphorylated MAVS (p-MAVS), limiting its signaling capacity

• This action restricts the RNA-sensing pathway's ability to trigger antiviral responses

• Similar to STING, KSHV ORF33 enhances PPM1G recruitment to MAVS

The dual targeting of both DNA and RNA sensing pathways positions PPM1G as a master regulator that balances innate immune responses. Experimental data from the study demonstrates that PPM1G knockout enhances responses to both DNA stimuli (poly(dA:dT)) and RNA stimuli (poly(I:C), SeV), with markedly increased phosphorylation of IRF3 in PPM1G-deficient cells .

This regulatory mechanism allows for fine-tuning of immune responses, preventing excessive inflammation while maintaining sufficient antiviral protection. Understanding these pathways is critical for developing strategies to modulate immune responses in infections and inflammatory diseases .

How can PPM1G antibodies be used in cancer research?

PPM1G antibodies can be valuable tools in cancer research, given the protein's roles in cellular regulation and immune signaling. Here are methodological approaches for applying PPM1G antibodies in cancer research:

• Expression profiling in cancer tissues:

  • The Prestige Antibody HPA035531 from Sigma-Aldrich has been used to examine PPM1G expression across 20 common cancer types as part of the Human Protein Atlas project

  • Use IHC with PPM1G antibodies (HPA035531, 1:1000-1:2500) to:

    • Map PPM1G expression patterns across different cancer types

    • Compare expression in tumor versus adjacent normal tissue

    • Correlate expression with clinical parameters and patient outcomes

• PPM1G and immune evasion in cancer:

  • Given PPM1G's role in negative regulation of innate immune signaling , it may contribute to tumor immune evasion

  • Research applications:

    • Compare PPM1G expression in tumors with varying levels of immune infiltration

    • Correlate PPM1G levels with expression of interferon-stimulated genes in tumor samples

    • Investigate whether PPM1G inhibition enhances anti-tumor immune responses

• PPM1G in virus-associated cancers:

  • The Science Advances study indicates that KSHV (a cancer-causing virus) hijacks PPM1G for immune evasion

  • Similar mechanisms may operate in other virus-associated cancers

  • Research approach:

    • Use PPM1G antibodies to study its role in virus-associated malignancies

    • Compare PPM1G activity in virus-positive versus virus-negative tumors

    • Investigate PPM1G-viral protein interactions in different cancer contexts

• Experimental techniques:

  • Immunohistochemistry:

    • Map PPM1G expression across tumor microenvironments

    • HPA035531 antibody is recommended at 1:1000-1:2500 dilution for IHC

  • Western blotting:

    • Compare PPM1G protein levels across cancer cell lines

    • Recommended antibody concentrations: 0.04-0.4 μg/mL for HPA035531

  • Immunoprecipitation:

    • Identify cancer-specific PPM1G protein interactions

    • ab70794 is recommended at 3 μg per mg of lysate

These approaches can help elucidate the complex roles of PPM1G in cancer biology and potentially identify new therapeutic strategies targeting this phosphatase or its regulatory pathways .

What are the considerations for using PPM1G antibodies in cell stress response studies?

Given PPM1G's involvement in cellular stress responses, PPM1G antibodies can be valuable tools for studying these processes. Here are methodological considerations for using PPM1G antibodies in stress response research:

• Experimental stress models and antibody applications:

• PPM1G in antiviral stress responses:

  • From the Science Advances study, PPM1G restricts cytosolic DNA and RNA sensing pathways

  • This function balances the intensity of antiviral responses

  • Research applications:

    • Use PPM1G antibodies to track changes in PPM1G localization during viral infection

    • Monitor PPM1G's association with stress response components

    • Compare PPM1G activity in cells exposed to different stressors

• Cellular localization during stress:

  • Stress often triggers protein relocalization

  • Immunofluorescence approach:

    • Use HPA035531 for IF (0.25-2 μg/mL)

    • Track PPM1G movement between cellular compartments during stress

    • Co-stain with markers of stress-induced structures

• Integration with stress response pathways:

  • PPM1G may interact with key stress response regulators

  • Methodological approach:

    • Co-immunoprecipitation using PPM1G antibodies:

      • ab70794 is validated for IP at 3 μg/mg lysate

      • Identify stress-specific PPM1G binding partners

    • Western blotting to analyze pathway components:

      • Use appropriate PPM1G antibodies to correlate expression with stress markers

      • Monitor phosphorylation status of pathway components

• Controls and validation:

  • Include appropriate controls for each stress condition

  • Use PPM1G knockdown or knockout cells to validate specificity

  • Time-course analyses may be critical as stress responses are often dynamic

These methodological approaches provide a framework for investigating PPM1G's roles in cellular stress responses using PPM1G antibodies, potentially leading to new insights into stress response mechanisms and their dysregulation in disease .