ITCH Antibody

What is ITCH protein and what cellular functions does it regulate?

ITCH is an E3 ubiquitin ligase that plays a fundamental role in regulating protein turnover and degradation within cells. This protein specifically targets a diverse range of proteins for degradation, including transcription factors, signaling molecules, and membrane receptors, by recognizing specific motifs or domains in these proteins and catalyzing the attachment of ubiquitin chains . ITCH functions primarily as a negative regulator of immune responses by promoting the degradation of proteins involved in T cell activation and cytokine signaling pathways . Research has demonstrated that ITCH prevents autoimmunity in both humans and mice, with loss-of-function mutations in the ITCH gene associated with severe autoimmune conditions characterized by elevated antibody production .

How are ITCH antibodies typically generated for research applications?

ITCH recombinant monoclonal antibodies are produced using sophisticated recombinant DNA technology through a multi-step process. Initially, the gene encoding the ITCH monoclonal antibody is synthesized by sequencing the cDNA of ITCH antibody-producing hybridomas . These hybridomas are generated by fusing myeloma cells with B cells isolated from animals that have been immunized with synthesized peptides derived from human ITCH protein . The synthesized gene is subsequently incorporated into an expression vector and transfected into host cells for cultivation. The resulting ITCH recombinant monoclonal antibodies are then purified from the cell culture supernatant using affinity chromatography techniques to ensure high specificity and purity for research applications .

What are the common applications of ITCH antibodies in laboratory research?

ITCH antibodies serve multiple critical functions in immunological and molecular biology research. They are primarily employed in Western blotting (WB) applications at recommended dilutions ranging from 1:500 to 1:5000, allowing researchers to detect and quantify ITCH protein expression in various cell and tissue samples . Additionally, ITCH antibodies are valuable tools for immunohistochemistry (IHC) applications, enabling the visualization of ITCH protein localization within cellular compartments and tissues . Enzyme-linked immunosorbent assays (ELISA) utilizing ITCH antibodies provide quantitative measurement of ITCH protein levels . In flow cytometry applications, these antibodies facilitate the analysis of ITCH expression in specific cell populations, particularly in research examining B cell activation and differentiation processes .

How can researchers effectively use ITCH antibodies to study B cell responses?

Researchers investigating B cell responses should implement a multi-parametric approach when utilizing ITCH antibodies. Flow cytometry represents the gold standard methodology, requiring careful sample preparation and antibody combinations. Cells should first be isolated and stained with viability dye, then pre-treated with anti-CD16/CD32 (Fc Block) to prevent non-specific binding . For comprehensive B cell subset analysis, ITCH antibody staining should be combined with markers including CD19, IgD, IgM, CD21/35, CD23, GL-7, CD38, and CD138 . For germinal center B cells specifically, a GL-7+CD38- gating strategy alongside ITCH staining yields optimal results .

For phosphorylation studies examining ITCH's impact on mTORC1 signaling, cells should undergo a specialized fixation protocol: 4% paraformaldehyde fixation at 37°C for 10 minutes, followed by 90% methanol fixation for 20 minutes on ice . When staining for phosphorylated S6 (P-S6), anti-P-S6-biotin antibody should be applied for 1 hour at room temperature, followed by fluorochrome-conjugated streptavidin . This approach allows researchers to correlate ITCH expression with mTORC1 activity in B cell subsets.

What are the methodological considerations when designing experiments to investigate ITCH's role in B cell proliferation and metabolism?

When designing experiments to study ITCH's impact on B cell proliferation and metabolism, researchers should implement a comprehensive experimental framework. For proliferation assessment, cells should be labeled with CellTrace violet or similar proliferation dyes prior to activation . B cells can be stimulated through multiple pathways, including BCR activation (anti-IgM) and TLR9 engagement (CpG), with ITCH protein displaying regulatory effects on both pathways .

Metabolic studies require specialized approaches: glycolytic capacity should be measured using a Seahorse XF analyzer to determine extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in ITCH-sufficient versus ITCH-deficient B cells . For mTORC1 activation studies, researchers should examine phosphorylation of S6 (P-S6) as an established downstream target through flow cytometry or western blotting . Importantly, timing is critical - ITCH regulates mTORC1 activity within 2 hours of stimulation, suggesting early time points (0.5, 1, 2 hours) are essential for capturing ITCH's regulatory effects .

To isolate ITCH's B cell-intrinsic effects from T cell influences, mixed bone marrow chimera models provide the optimal experimental system, allowing for competitive fitness assessment between wild-type and ITCH-deficient B cells within the same immunological environment .

What techniques are most effective for measuring ITCH-dependent changes in antibody production and quality?

Comprehensive analysis of ITCH-dependent changes in antibody production requires multiple complementary approaches. For total serum antibody quantification, ELISA plates should be coated with anti-mouse Ig overnight, while antigen-specific antibody responses require coating with appropriate antigen (e.g., NP-BSA for NP-specific responses) . To distinguish between high-affinity and low-affinity antibodies, researchers should employ differential coating strategies: high-affinity antibodies can be detected using low conjugation ratio antigens (5NP/BSA), while total antigen-specific antibodies are measured using high conjugation ratio antigens (30NP/BSA) . The difference between these measurements reveals the low-affinity antibody fraction.

For optimal ELISA protocol implementation, researchers should:

• Use serum dilutions between 1:5,000 and 1:125,000

• Incubate samples for 2 hours at room temperature

• Apply isotype-specific detection antibodies conjugated to horseradish peroxidase

• Develop with TMB substrate and stop with 1M phosphoric acid

• Measure absorbance at 450nm

For autoantibody detection specifically, anti-dsDNA ELISAs should use salmon sperm DNA (100 μg/ml) in sodium bicarbonate buffer, filtered through a 0.45-μm filter before coating plates .

What animal models are most appropriate for studying ITCH function in immune regulation?

Several animal models provide valuable insights into ITCH function, each with specific advantages for different research questions. Itch knockout (KO) mice represent the foundational model, displaying elevated serum antibody levels (particularly IgM and IgG1) and detectable autoantibodies against dsDNA . These mice exhibit normal numbers of preimmune B cell populations but elevated antigen-experienced B cells, making them ideal for studying ITCH's role in activated B cell responses .

For antigen-specific studies, the B1-8 transgenic mouse model crossed with Itch KO mice offers superior experimental control. These mice express the B1-8 heavy chain within the endogenous IgH locus, yielding a BCR that recognizes the NP antigen when paired with a lambda light chain . Importantly, this BCR can undergo class switching and somatic hypermutation in germinal centers, allowing researchers to track ITCH's influence on affinity maturation processes .

Mixed bone marrow chimeras provide the most rigorous approach for isolating B cell-intrinsic effects of ITCH. By reconstituting irradiated recipients with mixtures of wild-type and Itch-deficient bone marrow, researchers can directly compare the competitive fitness of ITCH-sufficient and ITCH-deficient B cells within the same animal . This approach has revealed that ITCH acts within B cells to limit naive and germinal center B cell numbers, with particularly strong effects on the latter population .

How should researchers interpret contradictory findings between ITCH roles in T cells versus B cells?

When navigating apparently contradictory findings regarding ITCH function in different lymphocyte populations, researchers must consider several factors. The most notable paradox involves Tfh cell differentiation - previous studies demonstrated that Itch-deficient T cells show defective T follicular helper (Tfh) cell differentiation in viral infection models . This finding appears contradictory to the elevated class-switched antibody levels observed in Itch-deficient mice, as Tfh cells are generally required for efficient IgG responses .

This apparent contradiction can be resolved by considering:

• Context-dependent T cell effects: Itch-deficient T cells may be specifically defective in becoming Tfh cells in response to Th1/IFN-γ-biased viral infections, but may still support spontaneous germinal centers in steady-state conditions .

• T cell phenotype heterogeneity: Evidence supports heterogeneity in Tfh cell phenotypes depending on the immune response type, suggesting Itch-deficient T cells may maintain certain Tfh functions .

• B cell gain-of-function effects: The potent gain-of-function in Itch-deficient B cells could drive increased antibody production with decreased reliance on Tfh cell help .

• Cell-type specific roles: ITCH likely has distinct molecular targets in different lymphocyte populations, explaining divergent phenotypic outcomes.

Researchers should employ cell-type specific conditional knockout models to definitively distinguish ITCH functions in different immune cell populations.

What are the clinical implications of ITCH-deficiency research findings for human autoimmune conditions?

Research on ITCH deficiency has significant translational implications for human autoimmune disorders. Loss-of-function mutations in the ITCH gene have been identified in humans with severe multi-faceted autoimmune disease accompanied by autoantibody production . These clinical manifestations align with the phenotypes observed in Itch-deficient mice, validating the relevance of these research models.

From a mechanistic perspective, ITCH's B cell-intrinsic role in limiting germinal center B cell accumulation, plasma cell generation, and antibody production provides new insight into autoimmune disease pathogenesis . The discovery that Itch-deficient B cells display increased proliferation and mTORC1 activity suggests that even subtle increases in these parameters can translate into substantial changes in antibody quantity and quality in vivo .

These findings have therapeutic implications:

• Interventions targeting GC B cell proliferation could significantly impact antibody responses in autoimmune conditions .

• Therapies designed to boost or suppress ITCH function specifically in germinal center B cells could effectively regulate antibody levels in clinical settings .

• The mTORC1 pathway represents a potential therapeutic target in ITCH-deficient patients, as inhibiting this pathway might counteract the hyperactivation observed in ITCH-deficient B cells .

• The connection between ITCH and metabolic fitness in B cells suggests that metabolic modulators could potentially normalize abnormal B cell responses in ITCH-deficient patients .

What are the optimal experimental conditions for using ITCH antibodies in immunoblotting applications?

For optimal immunoblotting results with ITCH antibodies, researchers should implement a carefully optimized protocol. ITCH antibodies perform most effectively in Western blotting at dilutions ranging from 1:500 for lower expression systems to 1:5000 for high-expression systems . Sample preparation is critical - proteins should be extracted using RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors if phosphorylation states are of interest.

For optimal separation of ITCH protein (approximately 113 kDa), 8-10% SDS-PAGE gels are recommended, with 25-50 μg of total protein loaded per lane. After electrophoresis, proteins should be transferred to PVDF membranes (preferred over nitrocellulose for higher binding capacity) using semi-dry or wet transfer systems. Blocking should be performed using 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

Primary ITCH antibody incubation should occur overnight at 4°C with gentle agitation, followed by extensive washing (3-5 times for 5-10 minutes each) with TBST buffer. HRP-conjugated secondary antibodies should be applied at 1:5000-1:10000 dilutions for 1 hour at room temperature. After additional washing steps, visualization can be performed using enhanced chemiluminescence (ECL) reagents with exposure times optimized based on signal intensity.

How can researchers effectively validate ITCH antibody specificity for their experimental systems?

Establishing ITCH antibody specificity is essential for experimental validity and requires a multi-layered validation approach. The gold standard validation method involves comparing antibody reactivity between wild-type samples and those derived from Itch knockout models . This approach provides definitive evidence of antibody specificity by demonstrating absence of signal in knockout samples.

For systems where knockout models are unavailable, RNA interference approaches offer an alternative validation strategy. siRNA or shRNA-mediated knockdown of ITCH should result in proportional reduction of antibody signal compared to control samples. Additionally, pre-adsorption controls can be employed, where ITCH antibody is pre-incubated with purified ITCH protein or immunizing peptide before application to samples - specific antibodies will show significantly reduced or eliminated signal.

Specificity can be further validated through immunoprecipitation followed by mass spectrometry to confirm ITCH is the predominant protein captured by the antibody. For research applications involving tissue samples, researchers should perform immunohistochemical staining with appropriate positive and negative control tissues, validating staining patterns against known ITCH expression profiles.

What immunohistochemical protocols yield optimal results when using ITCH antibodies on tissue sections?

Immunohistochemical detection of ITCH requires methodological precision to achieve optimal signal-to-noise ratios in tissue sections. For formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval is critical - heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) at 95-98°C for 20 minutes typically yields optimal results. For frozen sections, fixation with 4% paraformaldehyde for 10 minutes at room temperature is recommended.

Based on published methodologies, ITCH antibodies perform effectively in immunohistochemical applications when:

• Tissues are blocked with 5-10% normal serum from the same species as the secondary antibody

• Primary ITCH antibody is applied at optimized concentrations (typically 1:100 to 1:500) and incubated overnight at 4°C

• Detection is performed using biotin-streptavidin systems or polymer-based detection methods

• DAB (3,3'-diaminobenzidine) is used as the chromogen, producing brown staining for ITCH-positive regions

For scoring ITCH expression in tissues, a standardized system similar to that used in published studies is recommended: negative (0), weak (1), moderate (2), and strong (3) staining intensity . This scoring system allows for quantitative comparison between samples and treatment groups. In cancer studies specifically, ITCH typically displays cytoplasmic and occasionally membranous staining patterns, with invasive ductal carcinoma showing moderate to strong cytoplasmic and membranous staining .

How does ITCH regulate mTORC1 signaling in activated B cells?

ITCH plays a critical role in regulating the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in B cells, which influences multiple aspects of B cell biology. Research has demonstrated that ITCH limits mTORC1 activity within hours after B cell activation, indicating that ITCH regulates early activation pathways downstream of both the B cell receptor (BCR) and Toll-like receptor 9 (TLR9) . B cells lacking ITCH exhibit significantly increased phosphorylation of S6 (P-S6), a well-established downstream target of mTORC1, following stimulation .

The molecular mechanism through which ITCH regulates mTORC1 activation likely involves ITCH-mediated ubiquitination and subsequent degradation of positive regulators of the mTORC1 pathway. Proteomic profiling of Itch-deficient B cells revealed increased abundance of proteins activated by mTORC1, further supporting this regulatory relationship . This enhanced mTORC1 signaling contributes to the increased metabolic fitness observed in Itch-deficient B cells, including elevated glycolytic capacity that supports their enhanced proliferative potential .

Importantly, the ITCH-mTORC1 regulatory axis appears to be particularly significant in germinal center B cells in vivo, where Itch-deficient GC B cells exhibit enhanced proliferation and mTORC1 activity, contributing to their increased persistence and output of plasma cells . This finding identifies a novel negative regulatory pathway that limits B cell metabolism and proliferation, ultimately controlling antibody responses.

What is the relationship between ITCH deficiency and autoantibody production?

The relationship between ITCH deficiency and autoantibody production represents a clinically significant aspect of ITCH biology with direct relevance to human autoimmune disease. Both humans and mice with ITCH deficiency develop elevated serum antibody levels and autoantibodies, including anti-dsDNA antibodies that are characteristic of lupus-like autoimmune conditions . While these autoantibody levels are not as high as those seen in classical mouse models of lupus (e.g., New Zealand Black × New Zealand White F1 mice), they are significantly elevated compared to age-matched controls .

The mechanistic basis for autoantibody production in ITCH deficiency involves multiple factors:

• Enhanced B cell activation: ITCH-deficient B cells show increased activation and proliferation in response to various stimuli, leading to expanded populations of activated B cells that may include autoreactive clones .

• Dysregulated germinal center responses: ITCH deficiency results in increased numbers of germinal center B cells, which are critical sites for the generation of high-affinity antibodies including potential autoantibodies .

• Increased plasma cell output: The elevated numbers of plasma cells observed in ITCH-deficient mice contribute directly to increased antibody production, including autoantibodies .

• Reduced tolerance mechanisms: ITCH may play a role in B cell tolerance mechanisms that normally eliminate or suppress autoreactive B cells.

These findings suggest that therapeutic strategies targeting ITCH function or downstream pathways could potentially mitigate autoantibody production in autoimmune conditions.

How can researchers use ITCH antibodies to study the interaction between ITCH and other proteins in the ubiquitination pathway?

Investigating protein-protein interactions involving ITCH requires sophisticated methodological approaches that leverage ITCH antibodies in multiple experimental contexts. For co-immunoprecipitation studies, researchers should lyse cells in non-denaturing buffers (such as NP-40 or CHAPS-based buffers) that preserve protein-protein interactions. ITCH antibodies can be conjugated to agarose or magnetic beads for immunoprecipitation, followed by SDS-PAGE and immunoblotting for suspected interaction partners.

For in situ visualization of ITCH interactions with other proteins, proximity ligation assays (PLA) provide powerful capabilities. This technique requires primary antibodies against both ITCH and its potential binding partner, followed by species-specific secondary antibodies conjugated to complementary oligonucleotides. If the proteins are in close proximity (<40nm), the oligonucleotides can be ligated and amplified, producing a fluorescent signal that can be visualized by microscopy.

Researchers investigating the ubiquitination activity of ITCH should design in vitro ubiquitination assays containing:

• Purified recombinant ITCH protein

• E1 activating enzyme

• E2 conjugating enzyme

• Ubiquitin (which can be tagged for detection)

• ATP regeneration system

• Potential substrate proteins

After incubation, reaction products can be analyzed by immunoblotting with antibodies against the substrate protein to detect mobility shifts indicating ubiquitination, or with anti-ubiquitin antibodies following immunoprecipitation of the substrate.

For high-throughput identification of ITCH substrates, researchers can employ proteomics approaches comparing ubiquitinated protein profiles between wild-type and Itch-deficient cells, providing a comprehensive view of the ITCH-dependent ubiquitome.