ANAPC11 Antibody

What is ANAPC11 and what is its functional significance in cell cycle regulation?

ANAPC11 (Anaphase-promoting complex subunit 11) is a critical component of the anaphase-promoting complex/cyclosome (APC/C), which functions as a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle . ANAPC11 harbors a RING-H2 motif characterized by non-tandem His and Cys residues that coordinate zinc cations .

At the molecular level, ANAPC11 forms a heterodimer with the cullin-like subunit APC2, constituting the catalytic core of the APC/C complex . This core is critical for the APC/C's ability to catalyze ubiquitin chain elongation . The complex primarily mediates the formation of 'Lys-11'-linked polyubiquitin chains and, to a lesser extent, 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains, as well as branched 'Lys-11'/'Lys-48'-linked ubiquitin chains on target proteins .

ANAPC11 may function specifically to recruit E2 ubiquitin-conjugating enzymes to the complex, facilitating the ubiquitination of substrates targeted for degradation by the proteasome . This regulatory mechanism ensures that events of mitosis occur in the proper sequence through the degradation of anaphase inhibitors, mitotic cyclins, and spindle-associated proteins .

What are the different transcript variants and protein isoforms of ANAPC11, and how can they be specifically detected?

According to research on glioblastoma (GBM), 12 transcript variants of ANAPC11 have been recorded in the National Center for Biotechnology Information Reference Sequences database, encoding for 3 different protein isoforms . These can be categorized into:

• Group 1: Transcript variant 1, encoding isoform 1

• Group 2: Transcript variants 2-11, encoding isoform 2

• Group 3: Transcript variant 14, encoding isoform 4

Quantitative PCR (qPCR) studies have demonstrated that transcript variants 2-11 (encoding isoform 2) are predominant in glioma tissues, primary GBM cells, and classic GBM cell lines . For specific detection, researchers can design primers that target unique sequences in each group of transcript variants:

When using siRNAs for functional studies, researchers should consider that different siRNAs may target specific transcript variants. For example, siRNA-233 targets common sequences in all ANAPC11 transcript variants, while siRNA-454 and siRNA-496 spare group 2 transcripts (variants 2-11) . This distinction is important as functional effects may differ depending on which isoforms are knocked down.

How can I optimize Western blot protocols for accurate ANAPC11 detection?

For optimal Western blot detection of ANAPC11, follow these research-validated procedures:

• Sample Preparation:

  • Prepare protein lysates using RIPA lysis buffer supplemented with protease inhibitors

  • Quantify protein concentration using BCA Protein Assay Kit (Thermo Fisher)

  • Load equal amounts (20-30 μg) of protein into 10-12% SDS-PAGE gels

• Gel Electrophoresis and Transfer:

  • Run samples on 10% SDS-PAGE gels for optimal separation

  • Transfer proteins onto PVDF membranes (Roche)

  • Block membranes with 5% skim milk powder for 1 hour at room temperature

• Antibody Incubation:

  • Primary antibody: Use anti-ANAPC11 antibody at 1:1000 dilution

  • Incubate with primary antibody overnight at 4°C

  • Recommended antibodies include:

    • Anti-ANAPC11 (D1E7Q) Rabbit mAb (#14090, CST)

    • Anti-Apc11 ANAPC11 Antibody (A08555, Boster Bio) at 1 μg/mL

• Detection and Visualization:

  • Use HRP-conjugated secondary antibodies appropriate for your primary antibody species

  • Expected molecular weight: approximately 10 kDa (Note: Some antibodies may detect at 39-40 kDa or 68 kDa, possibly due to post-translational modifications or detection of protein complexes)

  • Include positive controls such as HeLa cell lysates

• Troubleshooting:

  • If detecting multiple bands, validate specificity using ANAPC11 knockdown/knockout samples

  • For weak signals, increase protein loading or antibody concentration

  • Fresh preparation of samples is recommended to avoid protein degradation

What immunoprecipitation techniques are effective for studying ANAPC11 protein interactions?

Immunoprecipitation (IP) is crucial for investigating ANAPC11's interactions with other proteins, particularly in understanding its role in ubiquitination pathways. Based on published research protocols:

• Standard Co-immunoprecipitation:

  • Lyse cells in non-denaturing lysis buffer containing protease inhibitors

  • Clear lysates by centrifugation (14,000g, 10 min, 4°C)

  • Incubate 500-1000 μg of protein with 1-5 μg anti-ANAPC11 antibody overnight at 4°C

  • Add protein A/G magnetic beads and incubate for 2-4 hours

  • Wash beads 3-5 times with washing buffer

  • Elute bound proteins with SDS sample buffer for Western blot analysis

• GST Pulldown Assay (for studying direct interactions):

  • Insert ANAPC11 coding sequence into a pGEX-6P-3 vector

  • Transform into E. coli and induce expression with 1 mM IPTG

  • Purify GST-ANAPC11 fusion protein using glutathione Sepharose beads

  • Incubate the fusion protein with cell lysates (e.g., T24 or UM-UC-3)

  • Analyze eluted complexes by Coomassie blue staining and Western blotting

• Ubiquitination Assays:

  • Treat cells with proteasome inhibitor MG132 (20 μM for 48h) to accumulate ubiquitinated proteins

  • Perform IP with anti-FOXO3 or anti-ANAPC11 antibody

  • Detect ubiquitinated proteins using anti-ubiquitin antibody (YM3636, ImmunoWay)

  • For cycloheximide (CHX) chase assays to study protein stability, add CHX to a final concentration of 30 μg/mL and collect cells at indicated time points

• Mass Spectrometry-Coupled IP:

  • Perform IP as described above

  • Separate eluted proteins on SDS-PAGE

  • Excise gel bands and perform tryptic digestion

  • Analyze peptides by LC-MS/MS to identify ANAPC11-interacting proteins

This approach has successfully identified FOXO3 as an ANAPC11 substrate in urothelial bladder cancer research .

What immunohistochemical protocols yield optimal results for ANAPC11 detection in tissue specimens?

For accurate immunohistochemical (IHC) detection of ANAPC11 in tissue specimens, researchers should follow these optimized protocols:

• Sample Preparation:

  • Fix tissues in 10% neutral-buffered formalin

  • Process and embed in paraffin

  • Section tissues at 4-5 μm thickness

• Antigen Retrieval:

  • Deparaffinize and rehydrate sections

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Maintain at 95-98°C for 15-20 minutes, then cool to room temperature

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block nonspecific binding with 5% normal serum

  • Primary antibodies:

    • Anti-ANAPC11 (NBP1-78050, Novus Biologicals)

    • Incubate overnight at 4°C with optimized dilution (typically 1:100-1:500)

• Detection System:

  • Apply HRP-conjugated secondary antibody

  • Develop with diaminobenzidine (DAB)

  • Counterstain with hematoxylin

• Scoring and Analysis:

  • Score ANAPC11 expression based on staining intensity and percentage of positive cells

  • For clinical correlation studies, divide samples into "high expression" and "low expression" groups

  • Correlate with clinical parameters such as T stage, lymph node status, and patient outcomes

Research has shown that ANAPC11 expression correlates with clinicopathological features, with elevated expression significantly associated with high T stage (p=0.002), positive lymph node metastasis (p=0.004), and poor outcomes in UBC patients .

How do researchers validate the specificity of ANAPC11 antibodies?

Rigorous validation of antibody specificity is crucial for reliable ANAPC11 research. Best practices include:

• Genetic Validation:

  • Compare staining in control vs. ANAPC11 knockdown/knockout samples

  • Utilize siRNA-mediated knockdown with constructs like siRNA-233 which targets all ANAPC11 transcript variants

  • Use CRISPR-Cas9-mediated knockout of ANAPC11 using lentiCRISPR v2 backbone to generate complete loss-of-function models

• Expression System Validation:

  • Test antibody against recombinant ANAPC11 protein

  • Compare endogenous vs. overexpressed ANAPC11 (using pcDNA3.1 plasmid containing ANAPC11 ORF)

  • Verify size correspondence with the predicted molecular weight (approximately 10 kDa)

• Cross-Reactivity Testing:

  • Test antibody against lysates from multiple species when making cross-species claims

  • Perform peptide competition assays with the immunizing peptide to confirm binding specificity

  • Validate in multiple cell lines with known ANAPC11 expression levels

• Multiple Antibody Concordance:

  • Compare results using different antibodies targeting distinct epitopes of ANAPC11

  • Verify consistent expression patterns across techniques (WB, IHC, IF)

  • Corroborate protein detection with mRNA expression data from qPCR or RNA-seq

• Application-Specific Validation:

  • For Western blot: Check for specific band at expected molecular weight

  • For IHC/IF: Include positive control tissues (skeletal muscle, heart) and negative controls (omission of primary antibody)

  • For IP: Verify pull-down of interacting partners like APC2

What is the role of ANAPC11 in cancer progression and how might it serve as a therapeutic target?

Research has revealed ANAPC11's significant role in cancer progression, particularly in urothelial bladder cancer (UBC) and glioblastoma multiforme (GBM):

• Clinical Significance:

  • Elevated ANAPC11 expression correlates with:

    • High T stage in UBC (p=0.002)

    • Positive lymph node metastasis (p=0.004)

    • Poor patient outcomes

  • In GBM, higher ANAPC11 expression associates with:

    • Higher glioma grades

    • Worse patient survival (shown by immunohistochemistry and survival analysis)

• Cellular Mechanisms in Cancer:

  • UBC: ANAPC11 enhances proliferation and invasiveness of UBC cells

  • GBM: ANAPC11 knockdown:

    • Promotes exit from cell cycle

    • Inhibits proliferation

    • Induces neuronal differentiation (elongated cell processes)

    • Upregulates neuron-associated genes involved in axonogenesis and synapse formation

• Molecular Mechanisms:

  • FOXO3 Degradation Pathway: ANAPC11 increases ubiquitination of FOXO3 transcription factor, decreasing its stability

  • Downstream Effects:

    • Downregulation of p21 (cell cycle regulator)

    • Decreased expression of GULP1 (lipid metabolism molecule and androgen receptor signaling effector)

  • Cell Cycle Impact: ANAPC11 knockdown in GBM reduces expression of key cell cycle proteins including geminin, CDT1, cyclin E1, TK1, cyclin A2, cyclin B1, p-Histone H3, and p-CDC2

• Therapeutic Potential:

  • The ANAPC11-FOXO3 regulatory axis represents a novel therapeutic target

  • Potential approaches include:

    • Small molecule inhibitors targeting ANAPC11's E3 ligase activity

    • Disruption of ANAPC11-FOXO3 interaction

    • Stabilization of FOXO3 to counteract ANAPC11's effects

    • Differentiation-inducing treatment leveraging ANAPC11 inhibition in GBM

• Research Applications:

  • CRISPR-Cas9 knockout models have validated ANAPC11's oncogenic role in vivo

  • Transcript variant-specific targeting may be necessary for optimal therapeutic effects, as different variants show distinct functional impacts

This evidence collectively supports ANAPC11 as both a prognostic biomarker and potential therapeutic target in multiple cancer types.

How can CRISPR-Cas9 technology be effectively employed to study ANAPC11 function?

CRISPR-Cas9 technology offers powerful approaches for investigating ANAPC11 function through precise genetic manipulation:

• Complete Knockout Strategy:

  • Vector Construction:

    • Use lentiCRISPR v2 backbone following Zhang's protocol

    • Design sgRNAs targeting early exons of ANAPC11 for complete functional disruption

  • Delivery System:

    • Package with psPAX2 and pMD2.G plasmids for lentiviral production

    • Transfect into 293T cells for viral packaging

    • Infect target cells and select with puromycin

• Validation of Knockout Efficiency:

  • Genomic Validation: PCR and sequencing of the targeted region

  • Protein Validation: Western blot using anti-ANAPC11 antibodies

  • Functional Validation: Assess cell cycle progression, as ANAPC11 knockout should disrupt APC/C function

• In Vivo Applications:

  • Generate ANAPC11-knockout cell lines

  • Implant into appropriate animal models (e.g., subcutaneous or orthotopic xenografts)

  • Assess effects on tumor growth and lymph node metastasis

• Isoform-Specific Targeting:

  • Design sgRNAs targeting specific exons unique to particular transcript variants

  • This approach can distinguish functions of different ANAPC11 isoforms

  • Important consideration: Transcript variants 2-11 (encoding isoform 2) dominate in most tissues/cell lines

• Experimental Readouts:

  • Cell Proliferation: CCK8 colorimeter analysis, EdU assay, cell counting

  • Cell Morphology: Assess changes like the elongated processes observed in GBM cells

  • Gene Expression: RNA-seq to identify differentially expressed genes

  • Ubiquitination: Analyze changes in substrate (e.g., FOXO3) ubiquitination patterns

• Rescue Experiments:

  • Reintroduce ANAPC11 (or specific isoforms) using expression vectors resistant to the sgRNA

  • Determine whether phenotypic changes can be reversed

  • Test mutant versions of ANAPC11 to identify critical functional domains

This comprehensive CRISPR-based approach has successfully demonstrated ANAPC11's role in promoting cancer cell growth and lymph node metastasis in UBC models and revealed its involvement in cell cycle regulation and differentiation in GBM .

What is the mechanism of ANAPC11-mediated FOXO3 degradation and what are its downstream consequences?

The ANAPC11-FOXO3 regulatory axis represents a crucial mechanism in cancer progression, particularly in urothelial bladder cancer (UBC):

• Biochemical Mechanism:

  • ANAPC11, as part of the APC/C complex, acts as an E3 ubiquitin ligase

  • It increases the ubiquitination level of FOXO3 protein

  • This ubiquitination targets FOXO3 for proteasomal degradation

  • The decreased stability of FOXO3 protein reduces its cellular levels and transcriptional activity

• Experimental Validation:

  • Immunoprecipitation coupled with mass spectrometry identified FOXO3 as an ANAPC11 interactant

  • Western blot analysis shows decreased FOXO3 protein levels with ANAPC11 overexpression

  • Proteasome inhibitor (MG132) treatment prevents FOXO3 degradation, confirming the ubiquitin-proteasome pathway's involvement

  • Cycloheximide (CHX) chase assays demonstrate reduced FOXO3 stability with ANAPC11 overexpression

• Downstream Molecular Consequences:

  • p21 Downregulation: FOXO3 normally activates p21 transcription; its degradation leads to decreased p21 levels

  • GULP1 Reduction: FOXO3 degradation results in decreased expression of GULP1, a downstream effector of androgen receptor signaling

  • These molecular changes collectively promote cell cycle progression and reduce cell cycle checkpoint control

• Cellular Phenotypic Effects:

  • Enhanced Proliferation: Reduced p21 levels alleviate cell cycle inhibition

  • Increased Invasiveness: ANAPC11 overexpression promotes aggressive phenotypes in UBC cells

  • Lymph Node Metastasis: ANAPC11 promotes UBC cell metastasis to lymph nodes in vivo

• Clinical Relevance:

  • ANAPC11 expression inversely correlates with FOXO3 protein levels in clinical samples

  • This regulatory axis is associated with poor clinical outcomes in UBC patients

  • The ANAPC11-FOXO3 axis represents a potential therapeutic target

This mechanistic understanding provides a foundation for developing targeted therapies that could either inhibit ANAPC11's E3 ligase activity or stabilize FOXO3 protein to counteract the oncogenic effects of this pathway.

How does ANAPC11 expression vary across different tissue types and what are the implications for research?

ANAPC11 exhibits distinct expression patterns across tissues, which has important implications for experimental design and interpretation:

• Normal Tissue Expression Profile:

  • High Expression: Skeletal muscle and heart

  • Moderate Expression: Brain, kidney, and liver

  • Low Expression: Colon, thymus, spleen, small intestine, placenta, lung, and peripheral blood leukocytes

• Expression in Pathological Conditions:

  • Cancer Upregulation:

    • Elevated in urothelial bladder cancer (UBC) compared to normal tissues

    • Upregulated in glioblastoma multiforme (GBM) compared to normal cells

    • Expression increases with higher grades of glioma

  • Clinical Correlations:

    • Higher expression associated with advanced T stage in UBC (p=0.002)

    • Positive correlation with lymph node metastasis (p=0.004)

    • Associated with worse outcomes in GBM patients

• Subcellular Localization:

  • Shows both cytoplasmic and nuclear localization

  • This dual localization may reflect different functional roles in cellular compartments

• Transcript Variant Distribution:

  • Transcript variants 2-11 (encoding isoform 2) dominate in:

    • Glioma tissues

    • Primary GBM cells

    • Classic GBM cell lines

  • This isoform preference should inform experimental design when studying ANAPC11

• Cell Cycle-Dependent Expression:

  • In oligodendrocytes: Enriched in G1-phase

  • In GBM cells: Enriched in S and G2/M phases

  • GBM cells with lower ANAPC11 expression show higher percentage in G1 phase

• Research Implications:

  • Tissue Selection: Choose appropriate positive controls (skeletal muscle, heart) for antibody validation

  • Background Consideration: Account for baseline tissue expression when comparing pathological samples

  • Cell Line Selection: Different cell lines may have varying baseline expression

  • Experimental Timing: Consider cell cycle phase when analyzing ANAPC11 function

  • Transcript Targeting: Design experiments to account for dominant isoforms in target tissues