rab4a Antibody

What is RAB4A and what cellular functions does it regulate?

RAB4A is a small GTPase belonging to the Ras superfamily that plays crucial roles in cellular trafficking processes. It functions primarily in the regulation of endosomal transport and vesicular trafficking pathways. RAB4A shares structural and biochemical properties with other members of the Ras gene superfamily . Recent research has demonstrated that RAB4A-directed endosome traffic significantly impacts pro-inflammatory mitochondrial metabolism in T cells through mechanisms involving mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation . Additionally, RAB4A interacts with multiple proteins including RAB11FIP1, RABEP1, ZFYVE20, RUFY1, SGSM1, SGSM2, and SGSM3, further highlighting its importance in intracellular transport networks .

What applications are validated for RAB4A antibodies in experimental research?

RAB4A antibodies have been validated for multiple experimental applications with specific recommended dilutions and protocols:

It's important to note that reagent optimization is sample-dependent, and titration in specific testing systems is recommended to obtain optimal results .

What are the key considerations for optimizing Western blot detection of RAB4A?

When optimizing Western blot protocols for RAB4A detection, researchers should consider:

How should RAB4A antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of RAB4A antibodies is essential for maintaining their reactivity and specificity:

How does the detection of active (GTP-bound) versus total RAB4A differ methodologically?

Detecting active (GTP-bound) versus total RAB4A requires distinct methodological approaches:

Total RAB4A detection:

• Western blotting using antibodies that recognize RAB4A regardless of nucleotide binding state (e.g., 10347-1-AP, PAB15420)

• Immunofluorescence to visualize total RAB4A distribution in fixed cells

• IHC to examine tissue expression patterns

Active RAB4A (GTP-bound) detection:

• Specialized anti-Rab4GTP monoclonal antibodies that specifically recognize the GTP-bound conformation

• Immunoprecipitation (IP) approaches to pull down active RAB4A and its binding partners

• Immunofluorescence to visualize active RAB4A localization

• Note that Western blot is not applicable for GTP-bound RAB4A detection since SDS denatures the GTPase, altering its conformation

When studying RAB4A activation state, researchers should consider using techniques that preserve protein conformation, such as native PAGE or specialized pulldown assays with GTP-binding domain proteins. The choice between these approaches depends on whether the research question focuses on RAB4A expression levels or its functional activity state.

What experimental approaches can reveal RAB4A's role in endosomal trafficking and metabolism?

To investigate RAB4A's role in endosomal trafficking and metabolism, consider these methodological approaches:

• Genetic manipulation strategies:

  • Expression of constitutively active RAB4A mutants (e.g., Q72L) to study gain-of-function effects on endosomal trafficking and metabolic pathways

  • RAB4A knockout or knockdown studies to assess loss-of-function consequences

  • Cell type-specific deletion models, such as T cell-specific Rab4A knockout mice

• Metabolic analysis methods:

  • Assessment of mitochondrial electron transport and flux through the tricarboxylic acid cycle in RAB4A-manipulated cells

  • Measurement of CD98 expression levels, which can be regulated by RAB4A and impacts amino acid transport

  • Kynurenine production assays to connect RAB4A activity with inflammatory metabolite generation

  • mTOR activation readouts, as RAB4A influences this key metabolic regulator

• Trafficking assays:

  • Endosomal recycling rate measurements using fluorescently labeled cargo proteins

  • Co-localization studies with endosomal markers to track RAB4A-positive compartments

  • Live-cell imaging to visualize RAB4A-dependent trafficking events

Recent research has demonstrated that RAB4A activation (via the Q72L mutation) specifically impacts CD98-dependent kynurenine production, mTOR activation, and mitochondrial metabolism in T cells, with consequences for immune cell lineage specification and autoimmune pathogenesis .

How can researchers troubleshoot conflicting results when studying RAB4A across different cell types?

When encountering conflicting results in RAB4A studies across different cell types, consider these methodological approaches:

• Cell type-specific RAB4A expression and function:

  • RAB4A expression levels vary between cell types, which may explain differential responses to manipulation

  • Quantify baseline RAB4A expression in your specific cell types via qPCR and Western blot

  • The research demonstrates that RAB4A has cell type-specific effects, expanding CD4+ T cells while depleting CD8+ T cells

• Antibody validation:

  • Ensure antibody specificity in each cell type being studied

  • Verify RAB4A detection in positive control cells such as HeLa, HEK-293T, mouse brain tissue, rat brain tissue, Neuro-2a, or PC-12 cells

  • Consider using multiple antibodies targeting different epitopes to confirm findings

• Experimental condition standardization:

  • Standardize cell culture conditions, as RAB4A function can be influenced by stress, nutrient availability, and cell density

  • Document passage number, as protein expression and trafficking pathways can change with cell culture duration

  • Consider the impact of serum factors on endosomal trafficking pathways

• Context-dependent protein interactions:

  • RAB4A interacts with multiple partners including RAB11FIP1, RABEP1, ZFYVE20, RUFY1, SGSM1, SGSM2, and SGSM3

  • These interaction partners may vary between cell types, affecting RAB4A function

  • Perform immunoprecipitation studies to determine cell type-specific RAB4A interactome

• Cell cycle considerations:

  • RAB4A localization and function are regulated by reversible phosphorylation-dephosphorylation during the cell cycle

  • Synchronize cells or document cell cycle status when comparing RAB4A function across experiments

What are the key considerations when designing studies to investigate RAB4A's role in autoimmune pathogenesis?

When investigating RAB4A's role in autoimmune pathogenesis, researchers should consider:

• Model system selection:

  • Mouse models with lupus-prone backgrounds (e.g., B6.TC) provide valuable insights into RAB4A's role in autoimmunity

  • Sex differences are important, as female B6.TC mice show elevated autoantibody production compared to males

  • Cell-specific RAB4A manipulation (e.g., T cell-specific knockout) helps isolate immune compartment contributions

• Readouts for autoimmune pathogenesis:

  • Measure autoantibody production (ANA, anti-ApoH, ACLA) to assess B cell effects

  • Analyze T cell subset distribution, particularly CD4+/CD8+ ratios and double-negative T cells

  • Evaluate organ-specific pathology, such as glomerulonephritis in lupus models

• Metabolic pathway assessment:

  • Connect RAB4A activity to CD98 expression, which influences amino acid transport

  • Measure kynurenine levels, which can spread inflammation through the bloodstream

  • Assess mTOR activation status, as it's a key downstream effector of RAB4A-driven autoimmunity

• Intervention strategies:

  • Test pharmacological mTOR blockade as a potential therapeutic approach

  • Consider targeting CD98 expression or function to mitigate RAB4A-driven effects

  • Evaluate temporal intervention windows to determine critical stages for RAB4A-driven pathogenesis

• Translational considerations:

  • Verify findings in human samples when possible

  • Correlate RAB4A expression/activity with clinical disease parameters

  • Consider biomarker potential of RAB4A-regulated pathways

The research indicates that Rab4A activation promotes CD4+ T cell expansion at the expense of CD8+ T cells, enhances B cell activation and plasma cell development, and increases autoantibody production in lupus-prone mice . These effects can be attenuated by Rab4A deletion in T cells or by pharmacological mTOR blockade .

How can researchers optimize immunofluorescence protocols for RAB4A detection?

For optimal immunofluorescence detection of RAB4A, consider these methodological recommendations:

• Fixation and permeabilization:

  • Use 4% paraformaldehyde for fixation to preserve cellular architecture

  • Test different permeabilization reagents (0.1-0.5% Triton X-100, 0.1% saponin, or methanol) as RAB4A is membrane-associated

  • Consider shorter fixation times (10-15 minutes) to preserve epitope accessibility

• Antibody selection and dilution:

  • Use antibodies validated for IF/ICC applications at recommended dilutions (1:50-1:500)

  • Consider performing an antibody titration to determine optimal concentration

  • Verify positive IF staining in control cells like HeLa cells

• Co-localization studies:

  • Include markers for early endosomes (EEA1), recycling endosomes, or other Rab proteins

  • Use confocal microscopy for precise co-localization assessment

  • Consider super-resolution microscopy for detailed endosomal compartment visualization

• Signal amplification:

  • If signal is weak, test signal amplification methods like tyramide signal amplification

  • Ensure appropriate secondary antibody selection with bright, photostable fluorophores

  • Optimize imaging parameters including exposure time, gain, and offset

• Controls:

  • Include negative controls (secondary antibody only, isotype control)

  • Use siRNA knockdown or knockout cells as specificity controls

  • Consider cells with known RAB4A expression patterns as positive controls

What are the critical parameters for successful immunohistochemical detection of RAB4A in tissue samples?

For optimal immunohistochemical detection of RAB4A in tissue samples:

• Tissue preparation and fixation:

  • Use freshly fixed tissues when possible, with controlled fixation times

  • Paraffin-embedded or frozen sections can be used, though protocol optimization differs

  • Section thickness of 4-6 μm is typically suitable for good antibody penetration

• Antigen retrieval methods:

  • Use TE buffer pH 9.0 as the recommended retrieval solution for RAB4A antibodies

  • Alternative approach: citrate buffer pH 6.0 may also be effective

  • Optimize retrieval time and temperature based on tissue type and fixation method

• Antibody dilution and incubation:

  • Use recommended dilutions of 1:50-1:500 as starting points

  • Perform titration experiments to determine optimal concentration

  • Consider longer incubation times (overnight at 4°C) for improved sensitivity

• Detection systems:

  • Polymer-based detection systems often provide better signal-to-noise ratio

  • DAB (3,3'-diaminobenzidine) provides a stable chromogenic signal

  • For multiplexing, consider fluorescent detection systems

• Positive control tissues:

  • Human heart tissue has been validated for positive RAB4A detection

  • Include known positive tissues in each staining batch

  • Consider cell type-specific expression patterns when interpreting results

• Counterstaining and mounting:

  • Light hematoxylin counterstain helps visualize tissue architecture

  • Use mounting media appropriate for long-term preservation

  • Document staining conditions precisely for reproducibility

How should researchers approach experimental design when studying RAB4A's interaction with specific binding partners?

When investigating RAB4A interactions with binding partners:

• Binding partner selection:

  • Focus on established RAB4A interactors such as RAB11FIP1, RABEP1, ZFYVE20, RUFY1, SGSM1, SGSM2, and SGSM3

  • Consider RAB4A's nucleotide-binding state, as interactions are often GTP-dependent

  • Use database resources to identify novel candidate interactors based on protein domains

• Co-immunoprecipitation approaches:

  • For active RAB4A (GTP-bound form), use anti-Rab4GTP-specific antibodies (1 μg for 1-2 mg total protein)

  • Include appropriate controls (IgG control, GTP/GDP loading controls)

  • Consider gentle lysis conditions to preserve membrane-associated complexes

• Nucleotide-dependent interaction studies:

  • Use non-hydrolyzable GTP analogs (GTPγS) to lock RAB4A in active conformation

  • Compare binding patterns between GDP-loaded (inactive) and GTP-loaded (active) states

  • Consider nucleotide-locked mutants (Q72L for GTP-bound, S22N for GDP-bound) as tools

• Localization studies:

  • Perform co-localization analysis by immunofluorescence

  • Consider live-cell imaging with fluorescently tagged proteins

  • Analyze subcellular fractionation to confirm compartment-specific interactions

• Functional validation:

  • Use siRNA/shRNA approaches to deplete binding partners

  • Assess effects on RAB4A-dependent trafficking or metabolic pathways

  • Design competition experiments with binding domain fragments

Recent research highlights that RAB4A interactions significantly impact T cell metabolism through effects on CD98 expression and mitochondrial function, with consequences for autoimmune disease progression .

What new methodologies are being developed to study RAB4A's role in mitochondrial metabolism?

Emerging approaches for investigating RAB4A's involvement in mitochondrial metabolism include:

• Mitochondrial functional assays:

  • Seahorse metabolic flux analysis to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in RAB4A-manipulated cells

  • Assessment of electron transport chain activity in relation to RAB4A activation state

  • Measurement of tricarboxylic acid cycle flux using stable isotope labeling approaches

• Mitophagy assessment:

  • Quantification of mitochondrial turnover using MitoTimer or mt-Keima reporters in RAB4A-modified cells

  • Analysis of mitophagy markers (PINK1, Parkin recruitment) in relation to RAB4A activity

  • Live imaging of mitochondrial dynamics in response to RAB4A manipulation

• Nutrient sensing pathway integration:

  • Investigation of RAB4A's impact on mTOR activation in different cellular compartments

  • Assessment of amino acid transport via CD98 expression regulation by RAB4A

  • Analysis of kynurenine production and signaling in response to RAB4A activity

• Single-cell metabolic profiling:

  • Application of single-cell metabolomics to identify RAB4A-dependent metabolic signatures

  • Correlation of RAB4A expression/activation with metabolic states at single-cell resolution

  • Integration with transcriptomic data to link RAB4A activity to metabolic gene programs

Recent research has established that RAB4A activation significantly impacts mitochondrial metabolism in T cells by regulating CD98 expression, kynurenine production, and mTOR activation . These metabolic changes contribute to T cell lineage specification and autoimmune pathogenesis, highlighting RAB4A as a multilevel regulator of immune cell metabolism .

How can researchers leverage RAB4A studies to develop potential therapeutic strategies for autoimmune diseases?

Based on recent findings about RAB4A's role in autoimmunity, researchers can explore these therapeutic strategies:

• Targeting RAB4A activity:

  • Development of small molecule inhibitors specific to RAB4A GTPase activity

  • Design of peptide-based inhibitors targeting RAB4A-effector interactions

  • Evaluation of nucleotide exchange inhibitors to prevent RAB4A activation

• Modulating downstream pathways:

  • mTOR inhibition approaches, which have shown efficacy in restraining RAB4A-driven autoimmunity

  • CD98 expression or function modulation to limit amino acid transport and inflammatory metabolism

  • Kynurenine pathway intervention to prevent pro-inflammatory metabolite accumulation

• Cell type-specific targeting:

  • T cell-targeted delivery systems for RAB4A-modulating therapeutics

  • Focus on restoring CD4+/CD8+ T cell balance disrupted by RAB4A hyperactivation

  • Prevention of double-negative T cell expansion associated with autoimmunity

• Biomarker development:

  • Assessment of RAB4A activation state in patient samples as a potential disease biomarker

  • Monitoring of CD98 expression levels as a readout of RAB4A activity

  • Measurement of serum kynurenine as an indicator of RAB4A-driven inflammatory metabolism

• Combination therapy approaches:

  • Testing RAB4A pathway inhibition alongside standard immunosuppressive agents

  • Sequential therapy targeting different aspects of RAB4A-driven autoimmunity

  • Personalized approaches based on individual RAB4A activation profiles

Research demonstrates that Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism, and T cell lineage skewing, and attenuate glomerulonephritis in lupus-prone mice . These findings provide a strong rationale for therapeutic targeting of this pathway in autoimmune diseases.