ANAPC5 Antibody

What is ANAPC5 and what cellular functions does it regulate?

ANAPC5 (Anaphase-Promoting Complex Protein 5) is a component of the multi-protein E3 ubiquitin ligase complex that plays dual roles in cell cycle regulation and immune signaling pathways . While traditionally known for its role in cell cycle progression through the anaphase-promoting complex, recent research has revealed that ANAPC5 interacts with interleukin-17 receptor subunits (IL-17RA and IL-17RC) and functions as a negative regulator of IL-17-mediated signaling . ANAPC5 associates with the C/EBPβ-activation domain (CBAD) in IL-17RA, a region previously linked to inhibitory signaling mechanisms . Additionally, ANAPC5 interacts with A20 (TNFAIP3), a deubiquitinase that serves as a negative feedback inhibitor of multiple inflammatory signaling pathways including TNFα, IL-1, and IL-17 . This interaction facilitates the inhibitory functions of A20 downstream of the IL-17 receptor complex.

What experimental applications are ANAPC5 antibodies validated for?

ANAPC5 antibodies have been validated for multiple experimental applications that enable comprehensive analysis of this protein across various research contexts. According to manufacturer validations, these applications include:

Researchers should note that optimal antibody dilutions may vary depending on sample type and experimental conditions, requiring optimization for each specific application . Additionally, validation data from manufacturers confirms reactivity with human ANAPC5, while some antibodies also cross-react with mouse and rat homologs .

How should ANAPC5 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of ANAPC5 antibodies is critical for maintaining their specificity and sensitivity. ANAPC5 antibodies are typically supplied as liquid formulations in PBS containing preservatives like sodium azide (0.02%) . For optimal stability, antibodies should be stored at -20°C for long-term preservation, where they remain stable for up to one year . For shorter periods (up to three months), storage at 4°C is acceptable .

Most importantly, researchers should avoid repeated freeze-thaw cycles, as these can lead to antibody degradation and reduced performance . To prevent this, it is recommended to aliquot the antibody into smaller volumes upon receipt before freezing. Additionally, antibodies should not be exposed to prolonged high temperatures or extreme pH conditions that could compromise their structural integrity and binding capacity .

How can researchers verify the specificity of ANAPC5 antibody for studies examining its interaction with IL-17 signaling pathways?

Verifying antibody specificity is critical when studying ANAPC5's role in IL-17 signaling networks. A comprehensive validation approach should include:

First, researchers should perform siRNA knockdown experiments targeting ANAPC5 followed by Western blot analysis to confirm that the detected band decreases or disappears following ANAPC5 silencing . In published studies, ST2 cells transfected with ANAPC5-specific siRNA demonstrated significantly reduced ANAPC5 expression levels compared to control siRNA, confirming both knockdown efficiency and antibody specificity .

Second, co-immunoprecipitation experiments can verify antibody specificity by demonstrating that the antibody captures protein complexes containing known ANAPC5 interaction partners. Research has shown that ANAPC5 co-immunoprecipitates with both IL-17RA and IL-17RC receptor subunits as well as with A20 (TNFAIP3) . These interactions can be confirmed by expressing tagged versions of these proteins (e.g., Myc- or HA-tagged) in systems like HEK293T cells, immunoprecipitating with the ANAPC5 antibody, and then blotting for the tagged proteins .

Finally, researchers can employ immunofluorescence microscopy to assess whether the antibody detects ANAPC5 in expected subcellular compartments, which can be compared with the localization of known interaction partners or with fluorescently tagged ANAPC5 expression constructs .

What are the key methodological considerations when using ANAPC5 antibodies to investigate its role in inhibiting IL-17 signaling?

When investigating ANAPC5's inhibitory role in IL-17 signaling, several methodological considerations are essential:

First, stimulation timing and conditions must be carefully optimized. Research has shown that following siRNA knockdown of ANAPC5, cells should be stimulated with IL-17 after sufficient time has elapsed for protein depletion (typically 48 hours post-transfection) . The concentration of IL-17 used for stimulation should be determined empirically, but published studies have demonstrated significant effects when measuring IL-6 induction as a readout of IL-17 signaling .

Second, appropriate controls are crucial: parallel experiments should include both a scrambled siRNA control and knockdown of established IL-17 signaling modulators (e.g., Act1 as a positive control for pathway inhibition, or A20 as another negative regulator) . This experimental design allows researchers to contextualize ANAPC5's relative contribution to IL-17 signal inhibition.

Third, researchers should assess multiple downstream readouts of IL-17 signaling beyond a single target gene. While IL-6 expression (both mRNA and protein) serves as a reliable indicator, examining other IL-17-responsive genes and signaling events provides a more comprehensive understanding of ANAPC5's inhibitory effects . This might include analysis of NF-κB activation, MAPK phosphorylation, and expression of additional IL-17 target genes.

Finally, the relationship between ANAPC5 and A20 should be investigated, as these proteins interact and likely cooperate in inhibiting IL-17 signaling . Co-immunoprecipitation experiments using both the long and short forms of ANAPC5 have revealed that ANAPC5, but not its related protein ANAPC7, interacts with A20, potentially explaining the differential effects of these proteins on IL-17 signaling .

How can domain-specific ANAPC5 antibodies be utilized to map its functional interactions with IL-17 receptor components?

Domain-specific ANAPC5 antibodies offer powerful tools for mapping the protein's functional interactions with IL-17 receptor components. Research has demonstrated that ANAPC5 interacts with specific domains of both IL-17RA and IL-17RC .

For IL-17RA interactions, researchers can employ a panel of truncation mutants spanning different regions of the IL-17RA cytoplasmic tail. Co-immunoprecipitation experiments have shown that ANAPC5 associates with the C-terminal domain of IL-17RA, corresponding to the C/EBPβ-activation domain (CBAD) . To map these interactions precisely, domain-specific antibodies that recognize distinct epitopes within ANAPC5 can be used in competitive binding assays to determine which regions of ANAPC5 are essential for IL-17RA association.

For IL-17RC interactions, similar approaches using truncation mutants of the IL-17RC cytoplasmic tail have revealed that ANAPC5 co-immunoprecipitates with all IL-17RC deletion mutants tested, suggesting that the interaction site is within the SEFIR domain of IL-17RC . Domain-specific antibodies can help confirm and further refine this mapping.

To comprehensively characterize these interactions, researchers should:

• Use epitope-specific ANAPC5 antibodies in co-immunoprecipitation experiments with various IL-17R mutants

• Perform competitive binding assays with recombinant ANAPC5 domains

• Complement antibody-based approaches with protein-protein interaction techniques like yeast two-hybrid screening, which was successfully used to identify ANAPC5 as an IL-17RC-interacting protein

This multi-faceted approach enables precise mapping of the molecular interfaces between ANAPC5 and IL-17 receptor components, facilitating a deeper understanding of the structural basis for ANAPC5's inhibitory function.

What optimization steps should be taken when using ANAPC5 antibodies for Western blot analysis?

Optimizing Western blot protocols for ANAPC5 detection requires attention to several key parameters:

First, sample preparation is critical. ANAPC5 is a component of a large multi-protein complex, so lysis conditions must preserve protein-protein interactions while efficiently extracting the protein. For co-immunoprecipitation studies examining ANAPC5's interactions with IL-17 receptors or A20, non-denaturing lysis buffers containing mild detergents such as NP-40 or Triton X-100 are recommended .

What controls should be included when performing immunoprecipitation studies with ANAPC5 antibodies?

Robust immunoprecipitation experiments with ANAPC5 antibodies require several essential controls:

First, input controls (typically 5-10% of the lysate used for immunoprecipitation) should always be included to verify the presence of target proteins in the starting material and to enable quantitative assessment of immunoprecipitation efficiency .

Second, isotype control antibodies matched to the host species and immunoglobulin subclass of the ANAPC5 antibody should be included to identify non-specific binding. For instance, if using a rabbit polyclonal ANAPC5 antibody, a non-specific rabbit IgG should be used as a negative control .

Third, when studying ANAPC5 interactions through co-immunoprecipitation, reciprocal immunoprecipitations should be performed. In published studies, researchers demonstrated ANAPC5 interaction with A20 by both immunoprecipitating with anti-ANAPC5 and blotting for A20, and conversely, immunoprecipitating with anti-A20 and blotting for ANAPC5 .

Fourth, specificity controls using cells where ANAPC5 expression has been knocked down via siRNA provide powerful validation. Studies have shown that ANAPC5 siRNA effectively reduces protein levels, confirming both knockdown efficiency and antibody specificity .

Finally, when examining interactions with transfected constructs, empty vector controls should be included to distinguish specific interactions from background binding. For example, when testing A20 interaction with ANAPC5, control plasmids were used to verify that co-immunoprecipitation occurred only with specific ANAPC5 constructs and not with control plasmids .

How can researchers optimize immunofluorescence protocols for ANAPC5 subcellular localization studies?

Optimizing immunofluorescence protocols for ANAPC5 localization requires careful attention to fixation, permeabilization, and antibody conditions:

Permeabilization methods significantly impact antibody accessibility to subcellular compartments. Since ANAPC5 functions both in the nucleus (cell cycle regulation) and cytoplasm (IL-17 signaling modulation), a balanced approach using 0.1-0.2% Triton X-100 is recommended as a starting point . For more specific compartmental analysis, researchers might consider digitonin (which preferentially permeabilizes plasma membranes) or stronger detergents for nuclear antigens.

Antibody dilution requires careful optimization, with typical ranges for immunofluorescence applications being more dilute than for Western blotting . Beginning with manufacturer recommendations and performing a dilution series (e.g., 1:100, 1:200, 1:500) allows identification of the optimal concentration that maximizes specific signal while minimizing background.

For co-localization studies investigating ANAPC5 interaction with IL-17 receptors or A20, dual labeling protocols should include appropriate controls for antibody cross-reactivity. When using antibodies from the same host species, sequential labeling protocols with intermediate blocking steps may be necessary to prevent cross-detection .

Finally, signal amplification methods such as tyramide signal amplification can be employed for detecting low-abundance interactions, particularly when examining ANAPC5's association with signaling components that may be transiently expressed or present at low levels .

What are common challenges and solutions when detecting ANAPC5 in co-immunoprecipitation experiments?

Co-immunoprecipitation experiments with ANAPC5 pose several common challenges that researchers should anticipate and address:

The first challenge is weak or absent signals in co-immunoprecipitation experiments. This may occur because ANAPC5 interactions with signaling components like IL-17 receptors or A20 can be transient or of relatively low affinity . To address this issue, researchers can employ crosslinking reagents (such as DSP or formaldehyde) prior to cell lysis to stabilize protein-protein interactions. Additionally, optimizing lysis conditions to use gentler detergents (0.5% NP-40 rather than stronger ionic detergents) can help preserve protein complexes.

Second, high background or non-specific binding can obscure genuine interactions. This is particularly challenging when studying ANAPC5, which functions as part of large multi-protein complexes . Solutions include increasing the stringency of wash buffers (gradually adding salt from 150mM to 300mM NaCl), pre-clearing lysates with protein A/G beads before immunoprecipitation, and using specific blocking agents (5% BSA) in wash buffers.

Third, competition between endogenous and exogenous (tagged) proteins can complicate analysis when using overexpression systems. Published studies have successfully addressed this by using cell lines with low endogenous expression of interaction partners or by employing siRNA knockdown of endogenous proteins while expressing siRNA-resistant tagged versions .

Fourth, the large size of ANAPC5-containing complexes can make some interactions difficult to detect due to steric hindrance limiting antibody access. Researchers have overcome this by using epitope-tagged constructs with the tag positioned to remain accessible within the complex, or by employing multiple antibodies recognizing different epitopes within ANAPC5 .

How can researchers address inconsistent results when examining ANAPC5's inhibitory effects on IL-17 signaling?

Inconsistent results when studying ANAPC5's inhibitory effects on IL-17 signaling can arise from several methodological variables:

First, cell type-specific effects may contribute to inconsistency. While studies have demonstrated ANAPC5's inhibitory role in ST2 cells, other cell types may exhibit different regulatory mechanisms depending on the expression levels of A20, ANAPC5, and other signaling components . Researchers should systematically compare multiple relevant cell types and potentially examine primary cells in addition to cell lines.

Second, the timing of analysis after IL-17 stimulation is critical. IL-17-induced signaling exhibits temporal dynamics, with some genes showing rapid induction while others respond more slowly . Studies demonstrating ANAPC5's inhibitory effects measured IL-6 expression, but the optimal timepoint for detecting this effect (24 hours post-stimulation) may not apply to all target genes . Researchers should conduct time-course experiments to identify the optimal window for observing ANAPC5-mediated inhibition.

Third, the efficiency of ANAPC5 knockdown can significantly impact results. Published studies verified knockdown efficiency by qPCR, confirming 70-80% reduction in ANAPC5 mRNA levels . Inconsistent results may stem from insufficient knockdown efficiency. Researchers should quantify knockdown at both mRNA and protein levels, potentially using multiple siRNA sequences to ensure robust silencing.

Fourth, the absence of appropriate positive controls can make interpretation difficult. Studies examining ANAPC5's inhibitory role included knockdown of established pathway components like Act1 (positive regulator) and A20 (negative regulator) as controls . Including these controls provides important benchmarks for contextualizing ANAPC5's relative contribution to signaling regulation.

Finally, the specific IL-17 concentration used for stimulation can affect results. Dose-response experiments can identify the optimal IL-17 concentration that best reveals ANAPC5's inhibitory effects, typically in the range of 100-200 ng/ml based on published studies .

What strategies can address cross-reactivity issues when using ANAPC5 antibodies in multi-protein complex studies?

ANAPC5 functions within multi-protein complexes, creating potential cross-reactivity challenges that require strategic approaches:

First, epitope mapping is essential to ensure antibody specificity. Commercial ANAPC5 antibodies are typically raised against specific peptide sequences, such as those "derived from internal of human ANAPC5" . When investigating multi-protein complexes, researchers should verify that the epitope recognized by their antibody doesn't share homology with other complex components, particularly other APC subunits like ANAPC7, which shares sequence homology with ANAPC5 .

Second, validation through multiple detection methods strengthens confidence in results. Published studies examining ANAPC5's interaction with IL-17 receptors and A20 confirmed findings using both co-immunoprecipitation and functional assays (knockdown effects on signaling) . This multi-method validation approach helps distinguish genuine interactions from potential cross-reactivity artifacts.

Third, recombinant protein controls can verify antibody specificity. Purified recombinant ANAPC5 can serve as a positive control in Western blots, while related proteins (particularly ANAPC7) can act as specificity controls to confirm that the antibody doesn't cross-react with similar proteins .

Fourth, knockout or knockdown validation provides powerful specificity confirmation. Studies demonstrated that siRNA targeting ANAPC5 reduced the signal detected by ANAPC5 antibodies, while knockdown of other APC complex components like ANAPC3 did not impact IL-17 signaling, supporting specific effects of ANAPC5 rather than cross-reactivity .

Finally, competitive blocking experiments using the immunizing peptide can confirm specificity. If an antibody was raised against a specific ANAPC5 peptide, pre-incubation with this peptide should abolish specific binding, providing a stringent test of antibody specificity in complex protein mixtures .

How might domain-specific ANAPC5 antibodies contribute to developing targeted therapeutics for IL-17-mediated inflammatory diseases?

Domain-specific ANAPC5 antibodies could play pivotal roles in developing novel therapeutics for IL-17-mediated inflammatory diseases through several research pathways:

First, epitope mapping antibodies can delineate the precise interaction surfaces between ANAPC5 and its binding partners. Research has shown that ANAPC5 binds to the CBAD domain of IL-17RA and interacts with A20, a key negative regulator of inflammatory signaling . Domain-specific antibodies that recognize distinct regions of ANAPC5 could identify the minimal interaction surfaces required for these protein-protein interactions, providing structural targets for therapeutic development.

Second, conformation-specific antibodies could differentiate between active and inactive states of ANAPC5. If ANAPC5's inhibitory function depends on specific conformational changes, antibodies recognizing these distinct states could serve as tools to screen for small molecules that stabilize the inhibitory conformation, potentially enhancing ANAPC5's negative regulatory effect on IL-17 signaling.

Third, function-blocking antibodies targeting specific ANAPC5 domains could modulate its inhibitory activity in experimental systems. For patients with hyperactive IL-17 responses driving pathology (as in psoriasis or inflammatory bowel disease), therapeutic approaches enhancing ANAPC5's inhibitory function could dampen excessive inflammation . Conversely, in contexts where enhanced IL-17 responses might be beneficial (certain infections), temporary neutralization of ANAPC5's inhibitory function might boost protective immunity.

Finally, domain-specific antibodies could facilitate high-throughput screening assays to identify compounds that modulate ANAPC5-A20 interactions. Since ANAPC5 interacts with A20 but not with other DUBs tested , compounds that enhance this specific interaction might augment the inhibitory effect on IL-17 signaling, providing a targeted approach to modulating inflammation without broadly suppressing immune function.

What novel techniques combining ANAPC5 antibodies with advanced imaging could reveal about its dynamic regulation of IL-17 signaling?

Advanced imaging approaches integrated with ANAPC5 antibody technology could transform our understanding of its dynamic regulatory functions:

Super-resolution microscopy techniques (STORM, PALM, or SIM) combined with domain-specific ANAPC5 antibodies could visualize the nanoscale organization of ANAPC5 within signaling complexes . This approach could reveal whether ANAPC5 forms discrete signaling nanodomains with IL-17 receptors and A20 at the plasma membrane or in endosomal compartments, providing spatial context for its inhibitory function.

Live-cell imaging using cell-permeable antibody fragments (Fabs) or intrabodies directed against ANAPC5 could track the dynamic recruitment of ANAPC5 to activated IL-17 receptor complexes in real-time. This would address whether ANAPC5 is constitutively associated with IL-17 receptors or recruited following ligand binding, helping establish the temporal sequence of events in ANAPC5-mediated inhibition.

Proximity ligation assays (PLA) with ANAPC5 antibodies could detect endogenous protein-protein interactions with exceptional sensitivity. This technique could confirm interactions between native ANAPC5 and IL-17 receptor components or A20 in primary cells at physiological expression levels, validating findings from overexpression systems .

FRET/FLIM microscopy using antibodies against ANAPC5 and its binding partners could measure interaction dynamics and conformational changes following IL-17 stimulation. This approach could determine whether A20 recruitment to IL-17 receptors depends on ANAPC5, as suggested by biochemical studies showing that ANAPC5, but not ANAPC7, co-immunoprecipitates with A20 .

Correlative light-electron microscopy (CLEM) employing ANAPC5 antibodies could bridge the resolution gap between fluorescence localization and ultrastructural context. This technique could reveal the microenvironmental features of ANAPC5-containing signaling complexes, potentially identifying specialized membrane domains or cytoskeletal associations relevant to IL-17 signal regulation.

How can researchers leverage ANAPC5 antibodies for high-throughput screening of compounds that modulate IL-17 signaling?

ANAPC5 antibodies can be strategically deployed in high-throughput screening platforms to discover modulators of IL-17 signaling:

AlphaScreen/AlphaLISA assays utilizing ANAPC5 antibodies can detect protein-protein interactions in a homogeneous format suitable for high-throughput screening. By coupling antibodies against ANAPC5 and A20 to donor and acceptor beads respectively, researchers could screen compound libraries for molecules that enhance or disrupt this interaction . Since ANAPC5-A20 binding appears critical for inhibiting IL-17 signaling, compounds affecting this interaction could serve as selective IL-17 pathway modulators.

Automated high-content imaging using fluorescently-labeled ANAPC5 antibodies could screen for compounds affecting ANAPC5 subcellular localization or its co-localization with IL-17 receptors . This approach could identify compounds that alter ANAPC5 recruitment to signaling complexes without directly disrupting protein-protein binding, potentially offering more nuanced modulation of pathway activity.

ELISA-based screening platforms using immobilized ANAPC5 antibodies could measure ANAPC5 protein levels or post-translational modifications following compound treatment. Since ANAPC5's inhibitory function may be regulated by modifications like ubiquitination or phosphorylation, compounds affecting these processes could modulate IL-17 signaling intensity .

Split-luciferase complementation assays incorporating ANAPC5 antibody-derived binding domains could monitor interaction dynamics in live cells. By fusing ANAPC5-binding antibody fragments and A20-binding fragments to complementary luciferase portions, researchers could develop a reporter system detecting compounds that modulate this interaction in a cellular context.

Antibody-based proteomics approaches could identify novel ANAPC5 interaction partners affected by screening hits. By immunoprecipitating ANAPC5 from cells treated with candidate compounds and analyzing co-precipitating proteins by mass spectrometry, researchers could uncover previously unknown components of ANAPC5-mediated inhibitory complexes, expanding the repertoire of potential therapeutic targets.