PXK Antibody

What is PXK and what are its key structural domains?

PXK (PX domain-containing protein kinase-like protein, also known as MONAKA or MONaKA) is a multimodular protein conserved in multicellular organisms from humans to flies. It possesses three key structural domains: a phox homology (PX) domain that binds phosphatidylinositol 3-phosphate, a protein kinase-like domain that may not display actual kinase activity, and a WASP homology 2 (WH2) domain that functions as an actin-binding domain . The PX domain is particularly important as it localizes PXK to endosomal membranes through binding to phosphatidylinositol 3-phosphate, making it critical for the protein's cellular functions .

What are the primary biological functions of PXK?

PXK serves several important cellular functions:

• It binds to and modulates brain Na,K-ATPase subunits ATP1B1 and ATP1B3, potentially participating in the regulation of electrical excitability and synaptic transmission

• It accelerates ligand-induced internalization and degradation of epidermal growth factor receptors (EGFR) through a mechanism requiring phosphatidylinositol 3-phosphate binding

• It enhances ubiquitination of EGFR induced by EGF stimulation

• It influences B-cell antigen receptor (BCR) internalization rates, which impacts B cell survival and cell fate decisions

How is PXK implicated in disease pathology?

Genome-wide association studies have identified variants in the PXK locus that confer risk for several humoral autoimmune diseases. Most notably, PXK variants have been associated with systemic lupus erythematosus (SLE), rheumatoid arthritis, and systemic sclerosis . Fine-mapping analysis has defined a large (257kb) common haplotype spanning the promoter through the 3' UTR of PXK that confers lupus risk in European ancestral populations . Functionally, individuals carrying the risk haplotype exhibit a decreased rate of BCR internalization, providing a mechanistic link between PXK variants and autoimmune disease pathogenesis .

What types of PXK antibodies are available for research, and how should I select the appropriate one?

Several types of PXK antibodies are available for research purposes:

• Polyclonal antibodies raised against synthetic peptides or recombinant fusion proteins containing sequences from human or mouse PXK

• Antibodies targeting specific domains (e.g., PX domain) or regions of the protein

When selecting a PXK antibody, researchers should consider:

• The species being studied (antibodies are available with reactivity to human, mouse, and rat PXK)

• The intended application (Western blot, immunohistochemistry, ELISA)

• The specific domain of interest (if studying a particular function of PXK)

• Validation data available for the antibody, including specificity and cross-reactivity information

How can I validate the specificity of a PXK antibody?

Validating a PXK antibody's specificity is crucial for obtaining reliable research results. Recommended validation approaches include:

• Western blot analysis using cell lysates from multiple cell lines to confirm detection of a band at the expected molecular weight (~65 kDa)

• Comparing reactivity in non-transfected cells versus cells transfected with PXK expression constructs

• Using siRNA-mediated knockdown of PXK to demonstrate reduced antibody signal

• Testing the antibody on tissue samples known to express PXK, such as brain tissue for immunohistochemistry

• Confirming that the antibody recognizes the appropriate domain or region of PXK based on peptide competition assays

For example, one commercially available PXK antibody (ab230514) has been validated by Western blot analysis of mouse brain lysate showing the expected 65 kDa band, as well as by immunohistochemistry on formalin-fixed, paraffin-embedded mouse brain cortex tissue .

What controls should be included when using PXK antibodies in experiments?

To ensure experimental rigor when using PXK antibodies, the following controls should be included:

• Positive controls: Cell lines or tissues known to express PXK (e.g., mouse brain for Western blot or IHC)

• Negative controls:

  • Primary antibody omission control

  • Non-specific IgG from the same species as the PXK antibody

  • Cells with siRNA-mediated PXK knockdown (as demonstrated in Vaughn et al., 2010)

• Loading controls: For Western blot, include housekeeping proteins like β-actin or GAPDH

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide to demonstrate specific blocking

• Transfection controls: Comparing antibody reactivity in cells overexpressing PXK versus non-transfected cells

What are the optimal conditions for using PXK antibodies in Western blot applications?

Based on the available research data, the following conditions are recommended for Western blot applications with PXK antibodies:

It's important to note that some researchers have generated custom antibodies against PXK by raising antisera against keyhole limpet hemocyanin-linked peptides corresponding to specific residues of human PXK . If commercial antibodies do not meet experimental needs, this approach provides an alternative.

How should PXK antibodies be used for immunohistochemistry on tissue sections?

For successful immunohistochemistry (IHC) with PXK antibodies:

• Tissue preparation: Formalin-fixed, paraffin-embedded tissues provide good results. Mouse brain cortex has been successfully used for PXK IHC

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended

• Antibody dilution: Typically 1:100 dilution works well for commercial PXK antibodies in IHC applications

• Detection system: Standard avidin-biotin complex or polymer-based detection systems are suitable

• Controls: Include positive control tissues (brain sections) and negative controls (primary antibody omission and non-specific IgG)

• Counterstaining: Hematoxylin provides good nuclear contrast without obscuring the PXK signal

Examine sections for PXK staining patterns associated with its known subcellular localization, particularly at the plasma membrane and in endosomal compartments.

What approaches can be used to study PXK localization in cells?

Several complementary approaches can be used to study PXK localization:

• Fluorescent fusion proteins: The PX domain of PXK can be subcloned into pEGFP-N1 or pEYFP-N1 vectors to express PXK as a fusion to the N-terminus of enhanced green or yellow fluorescent protein . This approach allows live-cell imaging of PXK dynamics.

• Immunofluorescence microscopy: Using PXK antibodies combined with co-staining for cellular markers such as:

  • Early endosome antigen 1 (EEA1) for early endosomes

  • Na,K-ATPase subunits (ATP1B1 and ATP1B3) to study interaction with these brain proteins

  • B-cell receptor components to study BCR internalization processes

• Subcellular fractionation: Isolating membrane fractions followed by Western blotting for PXK can confirm its association with specific cellular compartments

• Live-cell imaging with pH-sensitive probes: This can be particularly useful for studying the role of PXK in endocytosis and receptor trafficking

For example, previous research has shown that the PX domain of PXK localizes to endosomal membranes via binding to phosphatidylinositol 3-phosphate .

How can PXK antibodies be used to study B-cell receptor (BCR) internalization?

PXK antibodies are valuable tools for studying BCR internalization, which has been linked to lupus risk variants in the PXK locus . The following methodological approaches are recommended:

• Flow cytometry-based internalization assays:

  • Label surface BCRs with fluorescent anti-IgM antibodies

  • Measure the decrease in surface fluorescence over time after BCR stimulation

  • Compare cells from individuals with different PXK genotypes or cells with manipulated PXK expression

• Microscopy-based colocalization studies:

  • Use PXK antibodies together with markers for BCR and endocytic compartments

  • Quantify colocalization using appropriate imaging software

  • Track the temporal dynamics of BCR internalization in relation to PXK

• Biochemical fractionation:

  • Isolate membrane fractions at different time points after BCR stimulation

  • Analyze PXK and BCR components by Western blotting

  • Compare results between cells with different PXK genotypes or expression levels

Importantly, research has shown that individuals carrying the PXK risk haplotype exhibit a decreased rate of BCR internalization , suggesting a direct mechanism for how PXK variants contribute to autoimmunity.

What methods can be employed to study PXK's role in EGFR trafficking using PXK antibodies?

To investigate PXK's role in EGFR trafficking, researchers can employ the following methods:

• EGFR degradation assay:

  • Treat cells with EGF for various time periods (0-180 minutes)

  • Prepare cell lysates and analyze EGFR levels by Western blotting

  • Compare degradation kinetics between cells with normal, overexpressed, or silenced PXK levels

• EGFR internalization assay:

  • Surface-label EGFR with biotinylation reagents

  • Stimulate with EGF for various time periods

  • Measure remaining surface EGFR by streptavidin precipitation

  • Compare internalization rates in cells with manipulated PXK expression

• EGFR ubiquitination analysis:

  • Immunoprecipitate EGFR after EGF stimulation

  • Detect ubiquitination by Western blotting with anti-ubiquitin antibodies

  • Compare ubiquitination levels in cells with normal versus altered PXK expression

• Confocal microscopy:

  • Use fluorescently labeled EGF and immunofluorescence for PXK

  • Track colocalization during EGFR endocytosis

  • Employ acidic buffer washes to distinguish surface-bound from internalized receptors

Previous research has shown that PXK expression in COS7 cells accelerates ligand-induced internalization and degradation of EGFR through a mechanism requiring phosphatidylinositol 3-phosphate binding but independent of the WH2 domain .

How can researchers investigate the functional consequences of lupus-associated PXK variants?

To investigate how lupus-associated PXK variants affect cellular functions, researchers can employ these methodological approaches:

• Genotype-phenotype association studies:

  • Genotype subjects for PXK risk variants (e.g., rs6445972)

  • Measure BCR internalization rates in B cells from individuals with different genotypes

  • Correlate genotypes with clinical features or biomarkers of lupus

• CRISPR/Cas9 gene editing:

  • Introduce specific PXK variants into cell lines or primary cells

  • Compare receptor trafficking, cellular signaling, and immune responses between edited and unedited cells

  • Create isogenic cell lines differing only in PXK risk variants

• RNA interference:

  • Use siRNA to silence PXK expression in B cells or other relevant cell types

  • Three different siRNAs targeting different sites of the PXK gene have been validated for effective silencing

  • Target sequence example: 5′-GTTTAAGATCCCTACAAAG-3′

  • Assess effects on BCR internalization, B cell activation, and autoimmune phenotypes

• Transgenic mouse models:

  • Generate mice expressing human PXK risk variants

  • Analyze B cell development, activation, tolerance, and autoimmune phenotypes

  • Examine receptor trafficking in primary cells from these mice

Research has demonstrated that the strongest association with lupus risk was found at rs6445972 with P < 4.62 × 10^-10, OR 0.81 (0.75–0.86), and individuals carrying the risk haplotype exhibited decreased rates of BCR internalization .

What experimental approaches can assess how PXK interacts with the B-cell receptor signaling pathway?

To investigate PXK's interaction with BCR signaling pathways, researchers can employ these methodological approaches:

• Co-immunoprecipitation:

  • Immunoprecipitate PXK using validated antibodies

  • Probe for BCR components and signaling molecules in the precipitate

  • Perform reverse co-IP with BCR components and probe for PXK

  • Compare results before and after BCR stimulation

• Proximity ligation assay (PLA):

  • Use antibodies against PXK and BCR components

  • Visualize and quantify protein interactions in situ

  • Examine how interactions change upon BCR stimulation

• Calcium flux assays:

  • Load cells with calcium-sensitive dyes

  • Compare BCR-induced calcium responses in cells with normal versus altered PXK expression

  • Correlate findings with PXK genotype in primary B cells

• Phosphoproteomic analysis:

  • Stimulate BCR in cells with normal or altered PXK expression

  • Analyze phosphorylation changes in signaling molecules using mass spectrometry

  • Identify signaling pathways affected by PXK expression or genotype

• Live-cell imaging:

  • Use fluorescent fusion proteins to visualize PXK and BCR components

  • Track spatial and temporal dynamics during BCR stimulation

  • Correlate with endosomal markers to understand trafficking events

These approaches would help elucidate how PXK-mediated alterations in BCR internalization affect downstream signaling events that might contribute to autoimmunity.

How can PXK antibodies be used in combination with cellular uptake assays to predict antibody pharmacokinetics?

While not directly addressing PXK antibodies specifically, the search results describe methodologies for cell-based uptake assays that could be applied to study PXK or adapted for other research questions:

• Quantitative cellular uptake assay:

  • Use acid washing (confirmed to completely remove cell-bound antibodies) to distinguish internalized antibodies from cell-surface-bound antibodies

  • Monitor linear increases in uptake during incubation periods (up to 20 minutes)

  • Apply Michaelis-Menten modeling to analyze uptake kinetics

• Integration with pharmacokinetic (PK) prediction:

  • Determine in vitro kinetic parameters (Km and Vmax) from uptake assays

  • Combine with in vivo PK parameters from a minimal animal study

  • Create a 2-compartment model with Michaelis-Menten equation to predict PK profiles

This integrated approach follows the 3R (replacement, reduction, and refinement) principle by minimizing the number of animals needed for PK studies . While these methods were described for FcγRIIB-targeting antibodies, they could be adapted to study PXK-related uptake mechanisms or other receptor systems influenced by PXK.

What are the common technical challenges when using PXK antibodies, and how can they be addressed?

Based on general antibody usage principles and the available information about PXK antibodies, researchers might encounter these challenges:

• Low signal intensity in Western blots:

  • Optimize antibody concentration (try dilutions from 1:200 to 1:2000)

  • Increase protein loading (25-35 μg per lane is typically used)

  • Enhance detection with more sensitive ECL substrates

  • Extend exposure time beyond the standard 10 seconds

• Non-specific bands in Western blots:

  • Increase blocking time and concentration (3% nonfat dry milk in TBST is standard)

  • Try alternative blocking agents (BSA, commercial blockers)

  • Perform peptide competition to identify specific bands

  • Use PXK knockout or knockdown samples as negative controls

• Poor immunohistochemistry staining:

  • Optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer)

  • Adjust antibody dilution (1:100 dilution has been successful)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use amplification systems for signal enhancement

• Storage and stability issues:

  • Store antibodies at -20°C and avoid freeze/thaw cycles

  • Aliquot stock solutions to minimize freeze/thaw cycles

  • Use stabilizing proteins (BSA) in dilution buffers

  • Follow manufacturer recommendations for long-term storage

Proper validation with positive and negative controls is crucial to troubleshoot these issues effectively.

How can researchers effectively silence PXK expression for functional studies?

To effectively silence PXK expression for functional studies, researchers can use RNA interference approaches that have been successfully employed in previous research:

• siRNA transfection protocol:

  • Transfect cells with siRNA duplexes using Lipofectamine RNAi MAX reagent

  • Two days later, split cells into appropriate plate formats

  • Perform a second transfection with siRNA

  • Conduct functional assays 24 hours after the second transfection

• Validated siRNA sequences:

  • Three different siRNAs targeting different sites of the PXK gene have been tested and provided similar results regarding silencing efficiency

  • Example target sequence: 5′-GTTTAAGATCCCTACAAAG-3′

  • Use scrambled Stealth RNAi duplex as a negative control

• Verification of knockdown:

  • Prepare total RNA from transfected cells

  • Generate cDNA using appropriate kits (e.g., High Capacity cDNA Archive Kit)

  • Perform PCR to confirm silencing of PXK expression

  • Alternatively, verify knockdown by Western blot using validated PXK antibodies

• Functional readouts:

  • After confirming PXK knockdown, assess relevant cellular processes such as:

    • EGFR degradation or internalization rates

    • BCR internalization kinetics

    • Cell signaling responses

    • Interaction with Na,K-ATPase subunits