KBTBD7 Antibody, HRP conjugated

What is KBTBD7 and what cellular functions does it perform?

KBTBD7 functions as part of the CUL3(KBTBD6/7) E3 ubiquitin ligase complex, serving as a substrate adapter for the RAC1 guanine exchange factor (GEF) TIAM1. It mediates 'Lys-48' ubiquitination and proteasomal degradation of TIAM1, thereby regulating RAC1 signal transduction . This regulation impacts downstream biological processes including cytoskeleton organization, cell migration, and cell proliferation . Recent research also indicates that KBTBD7 enhances ubiquitin-dependent degradation of PTEN, thus activating EGFR/PI3K/AKT signaling pathways in NSCLC cells .

What applications are compatible with KBTBD7 antibody, HRP conjugated?

KBTBD7 antibody, HRP conjugated, can be used in multiple experimental applications including:

• Western blot (WB)

• Immunohistochemistry on paraffin-embedded tissues (IHC-P)

• Immunohistochemistry on frozen sections (IHC-F)

• Immunocytochemistry (ICC)

When using the antibody for Western blot applications, a dilution of 1/1000 has been successfully employed with human cell lines such as HL-60 (human promyelocytic leukemia cell line) .

What species reactivity has been confirmed for commercially available KBTBD7 antibodies?

Commercial KBTBD7 antibodies have been confirmed to react with human samples . Some antibodies also demonstrate reactivity with mouse and rat samples . Before application in non-human experimental models, validation experiments should be conducted to confirm cross-reactivity and specificity in the target species.

How should KBTBD7 antibody, HRP conjugated be stored to maintain reactivity?

To maintain optimal activity, KBTBD7 antibody with HRP conjugation should be stored according to manufacturer recommendations. Generally, this involves:

• Storage at -20°C for long-term preservation

• Avoidance of repeated freeze/thaw cycles that can degrade antibody quality and reduce sensitivity

• If working with aliquots, proper storage in single-use volumes to minimize freeze/thaw cycles

How does KBTBD7 expression correlate with clinicopathological features in cancer research?

Immunohistochemical studies of 104 paired NSCLC and peritumoral normal specimens revealed that KBTBD7 is highly expressed in NSCLC tissues compared to adjacent non-cancerous tissues . This overexpression positively correlates with several clinicopathological characteristics:

These findings suggest KBTBD7 may serve as a potential biomarker for NSCLC progression and could represent a therapeutic target .

What are the experimental considerations when performing ubiquitination assays to study KBTBD7's role in protein degradation?

When investigating KBTBD7's role in ubiquitin-dependent protein degradation (such as PTEN), researchers should consider the following methodological approach:

• Establish stable KBTBD7 knockdown cell lines (e.g., in NSCLC cell lines like A549 or H1299)

• Transfect cells with Ub-HA plasmid to enable detection of ubiquitination

• Pre-treat cells with proteasome inhibitor MG-132 (typically 12 hours before collection) to prevent degradation of ubiquitinated proteins

• Perform immunoprecipitation using antibodies against the target protein (e.g., anti-PTEN)

• Evaluate ubiquitination levels using anti-HA immunoblotting via Western blot analysis

This approach allows for assessment of how KBTBD7 affects the ubiquitination and subsequent degradation of target proteins.

How can researchers distinguish between transcriptional and post-translational effects of KBTBD7 on its target proteins?

To determine whether KBTBD7 regulates its target proteins at the transcriptional or post-translational level, researchers should implement a dual analysis approach:

• Transcriptional analysis: Perform RT-PCR to quantify mRNA levels of target proteins (e.g., PTEN) in both KBTBD7-suppressed and control cells. For example, studies show no change in PTEN mRNA levels when KBTBD7 is suppressed, suggesting regulation occurs post-transcriptionally .

• Protein interaction analysis: Conduct co-immunoprecipitation assays to verify direct protein-protein interactions between KBTBD7 and suspected targets .

• Protein degradation analysis: Use proteasome inhibitors (e.g., MG132) along with cycloheximide chase assays to assess protein stability and degradation rates.

• Ubiquitination assays: As detailed in question 2.2, these assays can confirm KBTBD7's role in ubiquitin-mediated protein degradation.

The combination of these techniques provides comprehensive evidence of KBTBD7's regulatory mechanisms.

What optimization strategies should be employed for immunohistochemistry using KBTBD7 antibody, HRP conjugated?

For optimal immunohistochemical detection of KBTBD7 in tissue specimens, researchers should consider the following protocol:

• Sample preparation:

  • Use tissue specimens sliced at approximately 4 μm thickness

  • Heat specimens at 70°C for 4–6 h before immunohistochemical staining

• Antigen retrieval:

  • Perform xylene deparaffinization followed by gradient alcohol hydration

  • Conduct heat-induced epitope retrieval using slightly boiling EDTA repair solution for 20 minutes

• Antibody incubation:

  • Use anti-KBTBD7 antibody at an optimized dilution (e.g., 1:100)

  • Incubate according to the manufacturer's recommended duration and temperature

• Scoring system implementation:

  • Assess staining intensity on a scale of 0-3: 0 (no staining), 1 (weak, light yellow), 2 (medium, yellow), 3 (strong, dark yellow/brown)

  • Evaluate the stained area on a scale of 1-4: 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), 4 (76%–100%)

  • Calculate final score by multiplying intensity and area scores (range: 0-12)

  • Consider specimens with scores >6 as KBTBD7-positive and those with scores ≤6 as KBTBD7-negative

What controls are essential when using KBTBD7 antibody in Western blot experiments?

When performing Western blot experiments with KBTBD7 antibody, HRP conjugated, the following controls should be included:

• Positive control: Include cell lines known to express KBTBD7, such as SK, A549, H1975, H1299, or HCC827 NSCLC cell lines, which demonstrate high expression levels compared to normal bronchial epithelial cells (HBE) .

• Negative control: Include cell lines with low or no KBTBD7 expression, or use siRNA/shRNA knockdown samples as negative controls.

• Loading control: Include detection of housekeeping proteins such as GAPDH to ensure equal loading across lanes.

• Antibody specificity control: Consider running a competition assay with the immunizing peptide to confirm specificity.

• Molecular weight marker: Include to confirm that the detected band corresponds to the expected molecular weight of KBTBD7.

What primer sequences are recommended for RT-PCR analysis of KBTBD7 expression?

For RT-PCR analysis of KBTBD7 expression, the following primer sequences have been successfully utilized:

KBTBD7:

• Forward: 5′-AGACGCCTTCGACCATCAC-3′

• Reverse: 5′-GAATTGAACCCATTCGGCTGA-3′

For comparison with related genes or targets, these additional primers may be useful:

PTEN:

• Forward: 5′-TGGATTCGACTTAGACTTGACCT-3′

• Reverse: 5′-GGTGGGTTATGGTCTTCAAAAGG-3′

EGFR:

• Forward: 5′-GGAGAACTGCCAGAAACTGACC-3′

• Reverse: 5′-GCCTACAGCACACTGGTTG-3′

GAPDH (reference gene):

• Forward: 5′-AGACGCCTTCGACCATCAC-3′

• Reverse: 5′-GAATTGAACCCATTCGGCTGA-3′

How can KBTBD7 antibodies be used to investigate cellular proliferation and invasion in cancer research?

KBTBD7 antibodies can be employed to investigate the relationship between KBTBD7 expression and cancer cell behavior through multiple experimental approaches:

• Expression analysis in clinical samples: Use immunohistochemistry with KBTBD7 antibody to correlate expression levels with tumor characteristics and patient outcomes .

• Functional studies in cell lines:

  • Create KBTBD7 knockdown cell lines and examine effects on proliferation (using CCK-8 and colony formation assays)

  • Assess invasion capabilities using Transwell assays

  • Examine expression of proliferation-related proteins (CCNE1, CDK4, P27) and invasion-related proteins (MMP-9, Claudin-1, Rock1, ZEB-1, E-cadherin) by Western blot following KBTBD7 suppression

• Signaling pathway analysis: Investigate how KBTBD7 affects key oncogenic signaling pathways, such as EGFR/PI3K/AKT, by examining phosphorylation status of pathway components after KBTBD7 modulation .

What are the considerations for subcellular localization studies of KBTBD7 using immunofluorescence?

When conducting immunofluorescence studies to determine KBTBD7's subcellular localization:

• Cell preparation: Both NSCLC cell lines and normal control cells (e.g., HBE) should be included for comparative analysis.

• Antibody selection: Use primary antibodies specific to KBTBD7 followed by fluorophore-conjugated secondary antibodies if the primary antibody is not directly conjugated.

• Co-localization markers: Include markers for specific cellular compartments (nuclear, cytoplasmic, membrane) to precisely determine localization.

• Microscopy parameters: Use confocal microscopy for higher resolution imaging to accurately determine subcellular distribution.

• Analysis and interpretation: Based on existing research, expect to observe predominantly cytoplasmic localization of KBTBD7 in both NSCLC and HBE cell lines .

How can KBTBD7's role in the ubiquitin-proteasome pathway be experimentally validated?

To verify KBTBD7's function in the ubiquitin-proteasome pathway:

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation to confirm KBTBD7's interaction with suspected substrate proteins (e.g., PTEN)

  • Use proximity ligation assays to visualize interactions in situ

• Ubiquitination assays:

  • Transfect cells with HA-tagged ubiquitin constructs

  • Pre-treat with proteasome inhibitors (MG-132)

  • Conduct immunoprecipitation of target proteins followed by anti-HA immunoblotting

• Protein stability analysis:

  • Perform cycloheximide chase assays in cells with normal or altered KBTBD7 expression

  • Monitor degradation rates of target proteins over time

• E3 ligase activity reconstitution:

  • Conduct in vitro ubiquitination assays with purified components including KBTBD7, CUL3, ubiquitin, E1, E2, and substrate proteins

This comprehensive approach provides strong evidence for KBTBD7's role as a component of the E3 ubiquitin ligase complex.

What are common issues when using KBTBD7 antibody, HRP conjugated in Western blotting and how can they be resolved?

When working with KBTBD7 antibody (HRP conjugated) in Western blotting, researchers may encounter the following challenges:

• High background signal:

  • Increase blocking time or concentration of blocking agent

  • Optimize antibody dilution (1/1000 has been successfully used for KBTBD7)

  • Include Tween-20 in wash buffers at appropriate concentration

  • Ensure membrane is fully covered during all incubation steps

• Weak or no signal:

  • Verify KBTBD7 expression in your sample (use positive control cell lines like A549, H1299)

  • Increase protein loading or antibody concentration

  • Optimize antigen retrieval if using tissue samples

  • Ensure protein transfer was successful using reversible stains

• Multiple bands or unexpected band sizes:

  • Verify antibody specificity with manufacturer

  • Consider post-translational modifications or splice variants

  • Include appropriate positive controls with known KBTBD7 expression

• Inconsistent results:

  • Standardize lysate preparation protocols

  • Avoid repeated freeze/thaw cycles of antibody

  • Maintain consistent incubation times and temperatures

What technical aspects should be considered when designing experiments to study KBTBD7's interaction with PTEN in cancer cells?

When investigating KBTBD7's interaction with PTEN and its implications in cancer:

• Cell line selection:

  • Use multiple NSCLC cell lines (e.g., A549, H1299) to ensure robustness of findings

  • Include cell lines with varying baseline PTEN expression levels

• KBTBD7 modulation approaches:

  • Employ both knockdown (siRNA, shRNA) and overexpression systems

  • Consider inducible systems for temporal control of expression

• Interaction validation:

  • Perform reciprocal co-immunoprecipitation (IP KBTBD7, blot PTEN and vice versa)

  • Use proximity ligation assays for in situ confirmation

  • Consider GST pull-down assays with recombinant proteins to confirm direct interaction

• Functional consequences:

  • Monitor PTEN protein levels and phosphorylation status of downstream targets (AKT)

  • Assess ubiquitination status of PTEN following proteasome inhibition

  • Examine phenotypic outcomes (proliferation, invasion) following PTEN rescue in KBTBD7-overexpressing cells

• Domain mapping:

  • Generate truncation or point mutation constructs to identify critical interaction domains

  • Assess how mutations affect KBTBD7-mediated PTEN degradation

These considerations ensure comprehensive characterization of the KBTBD7-PTEN regulatory axis in cancer contexts.