FBXL18 Antibody

What is FBXL18 and what cellular functions does it regulate?

FBXL18 (F-box and leucine-rich repeat protein 18) functions as a substrate recognition component of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex. It contains an F-box domain near its N-terminus, followed by several leucine-rich repeats and a transmembrane domain at its C-terminus . FBXL18 plays pivotal roles in:

• Protein ubiquitination and proteasomal degradation

• Cell cycle regulation

• Signal transduction

• Cancer progression, particularly in ovarian cancer and hepatocellular carcinoma

• K63-linked ubiquitination of target proteins such as AKT and RPS15A

FBXL18 is expressed in various tissues, with notable presence in gastroenterological and neural tissues .

What types of FBXL18 antibodies are currently available for research?

Several types of FBXL18 antibodies are available for research applications:

Most commercially available antibodies target either the full-length protein or specific epitopes within the N-terminal (aa 1-400) or C-terminal (aa 750 to C-terminus) regions .

How should I optimize Western blot protocols for FBXL18 detection?

For optimal Western blot results with FBXL18 antibodies:

• Sample preparation: Use RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitors (if studying phosphorylation).

• Protein amount: Load 20-40 μg of total protein per lane.

• Gel percentage: Use 8-10% SDS-PAGE gels as FBXL18 has a calculated molecular weight of 73 kDa .

• Transfer conditions: Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer with 20% methanol.

• Blocking: Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody incubation:

  • For mouse polyclonal antibodies: Use 1:500-1:1000 dilution in 5% BSA/TBST overnight at 4°C .

  • For rabbit polyclonal antibodies: Use 1:500 dilution as shown in Western blot analysis of PC-3M cell lysate .

• Detection: Use standard chemiluminescence methods. FBXL18 should appear as a band around 28.49 kDa (transfected) or at the calculated 73 kDa (endogenous) depending on the specific isoform and post-translational modifications .

What are the recommended protocols for immunohistochemistry with FBXL18 antibodies?

For optimal IHC staining of FBXL18:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissues sectioned at 4-6 μm thickness.

• Antigen retrieval:

  • Primary option: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Antibody dilutions:

  • For rabbit polyclonal antibodies: 1:20-1:200 dilution range, with 1:100 showing good results in human brain and lymphoid tissues

• Incubation: Incubate primary antibody overnight at 4°C in a humidified chamber.

• Detection system: Use an appropriate HRP-conjugated secondary antibody and DAB detection system.

• Counterstaining: Hematoxylin counterstaining provides optimal contrast as demonstrated in hepatic tissue analysis .

FBXL18 has been successfully detected in human brain tissue, lymphoid tissue, liver cancer tissue, and skin cancer tissue .

What are common technical issues when using FBXL18 antibodies and how can they be resolved?

How can I validate the specificity of my FBXL18 antibody?

To ensure FBXL18 antibody specificity:

• Positive controls: Use cell lines known to express FBXL18, such as OVCAR3, A2780, Huh7, HepG2, or A549 cells as demonstrated in published studies .

• Overexpression validation: Compare transfected cell lysate expressing FBXL18 (showing band at ~28.49 kDa) with non-transfected lysate as demonstrated in Western blot validations .

• RNA interference: Perform parallel experiments with FBXL18 knockdown using specific siRNAs or shRNAs as demonstrated in functional studies:

  • siRNAs that dramatically decreased FBXL18 mRNA and protein expression were validated by RT-qPCR and western blotting in ovarian cancer studies

  • shRNA validation options: TRCN0000155770, TRCN0000156053, or TRCN0000156441

• Peptide competition: Perform blocking experiments with the immunizing peptide to confirm specificity of binding.

• Cross-species validation: If working with mouse models, verify reactivity with mouse FBXL18 (antibodies with confirmed mouse reactivity are available ).

How can FBXL18 antibodies be used to study ubiquitination mechanisms?

FBXL18 antibodies can be instrumental in studying ubiquitination through these methodologies:

• Co-immunoprecipitation (Co-IP) assays:

  • Use anti-FBXL18 antibodies to precipitate protein complexes from cell lysates

  • Western blot for potential interacting partners (AKT, RPS15A)

  • For example, Co-IP assays demonstrated that FBXL18 strongly interacts with AKT in ovarian cancer cells

  • Similarly, FBXL18 was shown to interact with RPS15A in HCC cells

• Ubiquitination assays:

  • Immunoprecipitate target proteins (AKT, RPS15A) followed by immunoblotting with anti-ubiquitin antibodies

  • Specifically examine K63-linked ubiquitination using K63-specific antibodies

  • Studies revealed that FBXL18 promotes K63-linked AKT ubiquitination to activate AKT

  • Similarly, FBXL18 promotes K63-linked ubiquitination of RPS15A, enhancing its stability

• In vitro ubiquitination assays:

  • Set up reactions containing:

    • 50 mM Tris pH 7.6, 5 mM MgCl₂, 0.6 mM DTT, 2 mM ATP

    • 400 μM MG-132, 50 nM ubiquitin-activating enzyme

    • 0.5 μM UbcH5, 0.5 μM UbcH7, 2 μM ubiquitin

    • 1 μM ubiquitin aldehyde, 20 nM Cul1, 20 nM Rbx1, 20 nM Skp1

    • In vitro synthesized FBXL18 and target proteins

  • Detect ubiquitination by immunoblotting

How can FBXL18 antibodies be used to investigate cancer progression mechanisms?

FBXL18 antibodies can help elucidate cancer mechanisms through:

How should I interpret contradictory FBXL18 expression data across different cancer types?

When encountering contradictory FBXL18 expression patterns:

• Tissue-specific effects: FBXL18 acts as an oncogene in glioma, ovarian cancer, and hepatocellular carcinoma, but may have tumor suppressor roles in other cancers . Consider:

  • Different substrate availability across tissue types

  • Tissue-specific expression of competing F-box proteins

  • Variations in upstream regulators

• Methodological considerations:

  • Antibody specificity: Verify antibodies detect the same isoform

  • Detection methods: Compare mRNA (RT-qPCR) vs protein (Western blot, IHC) data

  • Sample types: Cell lines may differ from primary tissues

• Data integration approach:

  • Combine RNA-seq data (like TCGA datasets) with protein expression

  • Perform multivariate analysis controlling for clinical variables

  • Consider survival impact (Kaplan-Meier analysis showed FBXL18 expression was positively associated with poor survival in HCC patients, p<0.001)

What controls should be included when studying FBXL18's role in ubiquitination and protein degradation?

For rigorous ubiquitination studies, include these controls:

• Expression controls:

  • Empty vector controls alongside FBXL18 overexpression

  • Scrambled shRNA controls (parallel to FBXL18 knockdown)

  • F-box domain mutants that cannot form SCF complexes

• Specificity controls:

  • K63R ubiquitin mutants (cannot form K63 linkages)

  • Treatment with proteasome inhibitors (MG-132) to determine degradation dependence

  • Phosphorylation inhibitors (e.g., MK-2206 for AKT) to assess pathway dependence

• Interaction validation:

  • Reciprocal Co-IPs (immunoprecipitate AKT or RPS15A and blot for FBXL18)

  • Domain mapping with truncation mutants

  • In vitro binding assays with purified components

• Functional validation:

  • Proliferation/migration assays with FBXL18 manipulation

  • Rescue experiments with downstream components (e.g., constitutively active AKT)

  • Combined knockdown/overexpression of FBXL18 and its substrates

For example, MK-2206 treatment reversed the increase in proliferation and migration of ovarian cancer cells induced by FBXL18 overexpression, confirming AKT pathway dependence .

How can I use FBXL18 antibodies to explore potential therapeutic targets in cancer?

FBXL18 antibodies can facilitate therapeutic target discovery through:

• Target validation studies:

  • Immunoprecipitate FBXL18 complexes from cancer cell lines and identify novel interacting partners by mass spectrometry

  • Perform IHC on clinical samples to correlate FBXL18 expression with treatment response

  • Combine with phospho-proteomics to identify signaling nodes affected by FBXL18 manipulation

• Drug screening applications:

  • Develop cell-based assays using FBXL18 antibodies to measure protein levels after compound treatment

  • Screen for compounds that disrupt FBXL18-substrate interactions (e.g., FBXL18-AKT or FBXL18-RPS15A)

  • Monitor FBXL18 degradation as a potential therapeutic approach

• Combination therapy strategies:

  • Test FBXL18 inhibition in combination with existing therapies

  • For ovarian cancer: combine with AKT inhibitors like MK-2206

  • For hepatocellular carcinoma: combine with SMAD3 pathway inhibitors

  • Research has shown that the FBXL18/AKT axis plays a crucial role in ovarian cancer, indicating FBXL18 may be a valuable target for diagnosis and treatment

What methodologies can I use to study FBXL18's role in non-cancerous disease processes?

To investigate FBXL18 in non-cancerous conditions:

• Neurodegenerative disease models:

  • Evaluate FBXL18 expression in neuronal tissues using IHC with anti-FBXL18 antibodies

  • Explore potential interactions with neurodegeneration-related proteins

  • FBXL18 targets leucine-rich repeat kinase 2 for degradation by ubiquitination and attenuates neuronal toxicity, with mutations in FBXL18 linked to Parkinson's disease

• Inflammation and immune regulation:

  • Use transgenic mouse models (OE-FBXL18) to study inflammatory responses

  • Research showed FBXL18 induced more severe mild lobular, periportal, and pericentral inflammation in OE-FBXL18 mice compared to wild-type

  • Study the impact on inflammatory cytokine production and immune cell function

• Developmental processes:

  • Track FBXL18 expression during embryonic development using IHC

  • Investigate potential roles in organogenesis or cellular differentiation

  • Examine FBXL18's interaction with key developmental signaling pathways like SMAD3