RUBCN Antibody

What is RUBCN and why is it important in research?

RUBCN (Run domain Beclin-1-interacting and cysteine-rich domain-containing protein) functions primarily as a negative regulator of autophagy. It inhibits PIK3C3 activity and under basal conditions negatively regulates PI3K complex II (PI3KC3-C2) function in the autophagy pathway . RUBCN also negatively regulates endosome maturation and degradative endocytic trafficking, while impairing autophagosome maturation processes .

Research interest in RUBCN has grown significantly due to its involvement in multiple biological processes including:

• Autophagy regulation during fasting and aging

• B cell memory formation and immune responses

• Adipocyte lipid metabolism

• Exosome biogenesis and secretion

Studies have shown that RUBCN expression increases in aged tissues of worms, flies, and mice, suggesting it may be a signature of aging . Additionally, recent research has identified RUBCN as a key player in exosome biogenesis through its interaction with the WIPI protein family .

What are the known isoforms of RUBCN and their functional differences?

Research has identified multiple isoforms of RUBCN with distinct functions:

The shorter isoform, RUBCN100, has been found to enhance autophagy in B cells, while the longer RUBCN130 typically inhibits this process . Notably, mice lacking RUBCN130 or expressing RUBCN100 showed increased autophagy in B cells, which led to the generation of more memory B cells and suppressed plasmablast differentiation .

The balance between these two isoforms appears crucial for maintaining cellular equilibrium and controlling autophagy and mTORC1 activity . When designing experiments, researchers should consider which isoform(s) they wish to target and select antibodies accordingly.

What are the recommended applications for RUBCN antibodies?

Based on validated research protocols, RUBCN antibodies have been successfully employed in several applications:

Western Blotting (WB):
The most common application, allowing detection of different RUBCN isoforms based on molecular weight. Commercial antibodies like Abcam's ab156052 have been validated for WB applications with human and mouse samples .

Immunocytochemistry/Immunofluorescence (ICC/IF):
Used to visualize RUBCN localization and study its colocalization with autophagy markers like MAP1LC3. Research has shown that bafilomycin A1 treatment significantly increases the colocalization of GFP-RUBCN with MAP1LC3, an autophagosome marker .

Immunoprecipitation (IP):
Valuable for studying RUBCN's interactions with proteins like WIPI2 and components of the PtdIns3K complex. Studies have used domain-specific mutants to map interaction regions between RUBCN and its binding partners .

Immunoelectron Microscopy:
Provides ultrastructural localization of RUBCN, particularly useful for examining its association with multivesicular bodies (MVBs) and exosome formation .

When selecting antibodies for these applications, researchers should prioritize those validated for their specific experimental system and application.

What are the optimal protocols for detecting RUBCN by Western blotting?

Western blotting remains the gold standard for detecting and quantifying RUBCN protein levels. Based on published research protocols, the following methodology is recommended:

Sample Preparation:

• Lyse tissues or cells in an appropriate lysis buffer:

  • 50 mM Tris/HCl (pH 7.4)

  • 150 mM NaCl

  • 1 mM EDTA

  • 0.1% NP-40

  • Protease and phosphatase inhibitor cocktail

• Homogenize samples thoroughly using a homogenizer

• Centrifuge to remove debris and collect the supernatant

• Quantify protein concentration

Gel Electrophoresis and Transfer:

• Separate proteins using SDS-PAGE (5-12% polyacrylamide gels)

• Transfer proteins to PVDF membranes

Antibody Incubation:

• Block membranes with 5% skim milk in PBS containing 0.1% Tween 20

• Incubate with primary RUBCN antibody at appropriate dilution:

  • For mouse tissues: Cell Signaling Technology #8465 (1:500)

  • For Drosophila samples: custom dRubicon antibody (1:20,000)

• Use appropriate secondary antibody

• Include appropriate loading controls (β-actin at 1:8000 dilution)

Special Considerations:

• Different isoforms of RUBCN will appear at different molecular weights

• Treatment with lysosomal inhibitors like bafilomycin A1 may increase RUBCN levels, which could serve as a positive control

• For adipocyte studies, note that fasting causes substantial decreases in adipose RUBCN levels

How can I validate the specificity of a RUBCN antibody?

Rigorous validation is essential for obtaining reliable results with RUBCN antibodies. The following approaches are recommended:

Genetic Controls:

• Use RUBCN knockout (KO) models as negative controls

  • Rubicon-KO MEFs have been used successfully in multiple studies

  • CAG-Cre crossed with Rubicon flox mice can generate systemic Rubicon deletion models

  • Tissue-specific KO models (e.g., Nestin-Cre for brain-specific deletion) are valuable for tissue-specific studies

RNA Interference:

• Compare antibody signals in cells with and without RUBCN knockdown

• RNAi screening for RUBCN has been performed in human mesenchymal stem cells (hMSCs)

Overexpression Systems:

• Use cells overexpressing RUBCN (Rubicon-OE MEFs) as positive controls

• Compare signal intensity with non-transfected controls

Western Blot Analysis:

• Confirm that the antibody detects a protein of the expected molecular weight

• RUBCN130 should be approximately 130 kDa, while RUBCN100 should be around 100 kDa

• Check for the presence of non-specific bands

Multiple Antibody Comparison:

• When possible, compare results using antibodies targeting different epitopes

• Consistent results with different antibodies increase confidence in specificity

How can RUBCN antibodies be used to study autophagy regulation?

RUBCN's role as a negative regulator of autophagy makes its detection valuable for autophagy research. Effective experimental designs include:

Baseline Autophagy Assessment:

• Measure RUBCN levels alongside autophagy markers (LC3-II/I ratio, p62)

• Use Western blotting with antibodies for:

  • RUBCN (Cell Signalling Technology, #8465, 1:500)

  • LC3 (Cell Signalling Technology, #2755, 1:1000)

  • p62 (MBL, PM045, 1:1000)

• Correlate RUBCN expression with autophagy flux using lysosomal inhibitors

Autophagy Induction Studies:

• Track changes in RUBCN levels during autophagy-inducing conditions

• Studies have shown that fasting causes substantial decrease in adipose RUBCN levels

• Monitor autophagy markers in parallel to establish correlation

Immunofluorescence Approaches:

• Examine RUBCN colocalization with autophagy structures

• Bafilomycin A1 treatment significantly increases the colocalization of GFP-RUBCN with MAP1LC3

• Quantify colocalization using appropriate software and statistical methods

Isoform-Specific Analysis:

• Determine the relative abundance of RUBCN100 vs. RUBCN130

• Research has shown that RUBCN100 enhances autophagy in B cells, while RUBCN130 inhibits it

• Use isoform-specific detection methods to correlate with autophagy outcomes

When designing these experiments, researchers should include appropriate controls and consider the tissue/cell type-specific regulation of RUBCN and autophagy.

How can RUBCN antibodies be utilized to investigate exosome biogenesis?

Recent research has revealed RUBCN's critical role in exosome biogenesis through its interaction with WIPI proteins. To investigate this function:

Exosome Isolation and Analysis:

• Isolate exosomes from cell culture medium following knockdown or overexpression of RUBCN

• Analyze exosomal markers by Western blotting:

  • CD63 and Flotillin-1 levels in exosome fractions decrease with RUBCN knockdown

  • These markers increase in RUBCN-overexpressing cells

• Quantify exosome production using nanoparticle tracking analysis (NTA)

  • Studies show reduced numbers of small EVs in Rubicon-KO MEFs

Ultrastructural Analysis:

• Use electron microscopy to examine multivesicular body (MVB) formation

  • The number of MVBs decreases in RUBCN-deficient cells

• Employ immunoelectron microscopy to visualize:

  • RUBCN localization to MVBs

  • GFP-CD63-decorated intraluminal vesicles (ILVs)

Mechanistic Investigation:

• Study the RUBCN-WIPI axis using co-immunoprecipitation

  • The C-terminus and HCR domain of RUBCN are critical for WIPI2 interaction

• Investigate how RUBCN-WIPI interaction affects ESCRT machinery recruitment

  • WIPI2 interacts with multiple ESCRT components in a RUBCN-dependent manner

Rescue Experiments:

• Express wild-type or mutant RUBCN in knockout cells

• The ΔC mutant fails to rescue exosomal marker defects, indicating the importance of the C-terminus

• Quantify restoration of exosome production

These approaches enable comprehensive investigation of RUBCN's role in the complex process of exosome biogenesis.

What experimental approaches can be used to study the interaction between RUBCN and WIPI proteins?

The RUBCN-WIPI axis has emerged as a critical regulator of cellular processes including exosome formation. To study this interaction:

Domain Mapping:

• Use a series of RUBCN mutants lacking specific domains:

  • N-terminal serine-rich region (SR-N)

  • Coiled-coil domain (CCD)

  • C-terminal serine-rich region (SR-C)

  • Helix-coil-rich region (HCR)

  • C-terminal homology domain (RHD)

• Perform co-immunoprecipitation with each mutant

• Research has shown that the ΔC and ΔHCR mutants fail to pull down WIPI2

Co-Immunoprecipitation:

• Immunoprecipitate RUBCN and detect associated WIPI proteins

• Perform reciprocal experiments (pull down WIPI proteins and detect RUBCN)

• Include appropriate controls:

  • Input controls (portion of lysate before immunoprecipitation)

  • Negative controls (non-specific IgG or lysates from RUBCN knockout cells)

Cellular Localization:

• Visualize RUBCN and WIPI protein colocalization using immunofluorescence

• Quantify Rubicon-positive endosomes in control vs. WIPI2-deficient cells

  • Studies show these structures are reduced in WIPI2-deficient cells

Functional Analysis:

• Compare MVB formation in control vs. cells with disrupted RUBCN-WIPI interaction

• Analyze exosome production when the interaction is disrupted

• The number of MVBs containing GFP-CD63-decorated ILVs is reduced in RUBCN-deficient cells

These approaches provide complementary data to elucidate the RUBCN-WIPI interaction and its functional consequences.

How can RUBCN antibodies be used to track changes in autophagy during aging studies?

RUBCN expression increases with age and functions as a negative regulator of autophagy, making it an important marker in aging research:

Age-Related Expression Analysis:

• Compare RUBCN levels across age groups:

  • Young, middle-aged, and old animals

  • Multiple tissues from each age group

• Western blotting protocol:

  • Use lysis buffer: 50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, protease/phosphatase inhibitors

  • Rubicon antibody (Cell Signalling Technology, #8465, 1:500)

  • β-actin control (1:8000)

• Research has shown increased RUBCN expression in aged tissues of worms, flies, and mice

Autophagy Correlation:

• Simultaneously analyze autophagy markers alongside RUBCN:

  • LC3 (Cell Signalling Technology, #2755, 1:1000)

  • p62 (MBL, PM045, 1:1000)

• Calculate LC3-II/I ratios to assess autophagosome formation

• Determine correlation coefficients between RUBCN levels and autophagy markers

Intervention Studies:

• Include calorie restriction as a positive control for autophagy enhancement

  • Studies used a 40% calorie restriction protocol developed by Turturro et al.

• Examine how interventions affect the age-related increase in RUBCN expression

• Correlate RUBCN changes with functional outcomes

Tissue-Specific Analysis:

• Use tissue-specific RUBCN knockout models

  • Nestin-Cre mice for brain-specific deletion

• Compare autophagy markers and age-related changes with and without RUBCN

• Assess functional outcomes in different tissues

This systematic approach enables researchers to establish mechanistic links between RUBCN expression, autophagy decline, and aging phenotypes.

What are the technical challenges in detecting RUBCN in tissue samples versus cell culture?

Detecting RUBCN in tissues presents unique challenges compared to cell culture systems:

Tissue-Specific Extraction Protocols:

Species-Specific Considerations:

• Different species require validated antibodies:

  • Mouse: Cell Signalling Technology, #8465, 1:500

  • Drosophila: Custom antibody against C+EVPEEVHEKLQQAS peptide

• Consider raising species-specific antibodies for non-model organisms

Comparison with Cell Culture:

• Cell culture advantages:

  • More homogeneous samples

  • Easier manipulation (knockout, overexpression)

  • Cleaner background in immunofluorescence

• Cell culture limitations:

  • May not reflect in vivo complexity

  • Potential artifacts from immortalized cell lines

When transitioning between tissue and cell culture studies, researchers should validate RUBCN detection protocols for each experimental system and consider the physiological relevance of their findings.

What are the important controls when using RUBCN antibodies in immunofluorescence?

Robust controls are essential for reliable immunofluorescence experiments with RUBCN antibodies:

Specificity Controls:

• Genetic controls:

  • RUBCN knockout cells/tissues as negative controls

  • Cells overexpressing RUBCN as positive controls

• Antibody controls:

  • Primary antibody omission

  • Isotype control (non-specific IgG)

  • Multiple antibodies targeting different epitopes when possible

Technical Validation:

• Treatment response controls:

  • Bafilomycin A1 increases colocalization of GFP-RUBCN with MAP1LC3

  • This serves as a positive control for RUBCN-autophagosome association

• Subcellular marker controls:

  • Co-stain with established markers (e.g., MAP1LC3 for autophagosomes)

  • Include organelle markers to establish subcellular localization

Quantification Approach:

• Analyze multiple fields per sample (>10 recommended)

• Count sufficient cell numbers for statistical validity (>50 cells per condition)

• Use established colocalization algorithms

• Report appropriate statistical measures

When studying RUBCN's interaction with other proteins (e.g., WIPI2), include additional controls demonstrating the specificity of this interaction, such as domain mutants that disrupt binding.

How can I design experiments to study differential roles of RUBCN isoforms?

The distinct functions of RUBCN isoforms (RUBCN100 and RUBCN130) require careful experimental design:

Isoform Detection Strategy:

• Western blotting can distinguish isoforms by molecular weight

• Select antibodies recognizing epitopes present in both isoforms

• Validate using overexpression systems with individual isoforms

Functional Differentiation:

• B cell memory formation:

  • Mice lacking RUBCN130 generate more memory B cells

  • This process depends on enhanced autophagy

• Autophagy regulation:

  • RUBCN100 enhances autophagy in B cells

  • RUBCN130 inhibits autophagy

• Exosome biogenesis:

  • Compare exosome production in cells expressing specific isoforms

  • Analyze markers like CD63 and Flotillin-1

Model Systems:

• Genetic models:

  • Use mice lacking RUBCN130 or expressing RUBCN100

  • Create isoform-specific knockouts or knockins

• Cell culture:

  • Express individual isoforms in RUBCN-null backgrounds

  • Use inducible expression systems to control isoform levels

Interaction Analysis:

• Compare binding partners of each isoform

• Focus on known interactors like WIPI2 and components of the PtdIns3K complex

• Determine if isoforms differentially affect WIPI2's interaction with ESCRT components

The balance between RUBCN130 and RUBCN100 appears crucial for cellular homeostasis , making the ratio between isoforms an important experimental parameter to measure and manipulate.

What are the emerging research areas involving RUBCN antibodies?

Several promising research directions are emerging for RUBCN antibody applications:

• Aging and Neurodegeneration:
  RUBCN expression increases with age in diverse organisms, positioning it as a potential therapeutic target for age-related diseases . Researchers can use RUBCN antibodies to track expression changes in neurodegenerative disease models and evaluate interventions that modulate RUBCN levels.

• Immunometabolism:
  The involvement of RUBCN in both B cell memory formation and adipocyte metabolism during fasting points to its role at the intersection of immunity and metabolism. This creates opportunities for studying how metabolic changes influence immune function through RUBCN-mediated pathways.

• Extracellular Vesicle Biology:
  The discovery of RUBCN's role in exosome biogenesis through the RUBCN-WIPI axis opens new avenues for understanding intercellular communication. Researchers can use RUBCN antibodies to investigate how this protein regulates the cargo selection and production of exosomes in different physiological contexts.

• Therapeutic Development:
  As understanding of RUBCN's various functions expands, antibodies will be essential tools for validating it as a therapeutic target and evaluating the efficacy of interventions aimed at modulating its activity or expression.

RUBCN antibodies will continue to be critical tools for advancing our understanding of this multifunctional protein and its diverse roles in cellular homeostasis.

What best practices should researchers follow when publishing research using RUBCN antibodies?

To ensure reproducibility and reliability, researchers should adhere to these guidelines when publishing RUBCN antibody-based research:

• Detailed Antibody Documentation:

  • Provide complete antibody information: manufacturer, catalog number, lot number, RRID (Research Resource Identifier)

  • Specify the target epitope and species reactivity

  • Document validation experiments performed in your system

• Experimental Protocol Transparency:

  • Include detailed methods for sample preparation

  • Specify buffer compositions, antibody dilutions, and incubation conditions

  • For tissues, describe fixation methods and antigen retrieval protocols

• Control Documentation:

  • Show appropriate positive and negative controls

  • Include genetic controls when available (knockout, knockdown)

  • For overexpression studies, verify expression levels

• Image Acquisition Parameters:

  • Report microscope settings (exposure, gain, objective)

  • Describe image processing methods

  • Include scale bars on all micrographs

• Quantification Methods:

  • Detail statistical approaches for analyzing Western blots or immunofluorescence

  • Report sample sizes and biological replicates

  • Include raw data when possible