TNFRSF17 Antibody

What is TNFRSF17/BCMA and what is its biological significance?

TNFRSF17/BCMA is a member of the TNF receptor superfamily that plays a crucial role in B cell development and function. It is a type III membrane protein containing one extracellular cysteine-rich domain that shares high homology with TACI. Human BCMA is a 184 amino acid protein consisting of a 54 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a 107 amino acid intracellular domain .

BCMA primarily binds two ligands - APRIL and BAFF - which stimulate IgM production in peripheral blood B cells and enhance survival of cultured B cells . While some BCMA expression occurs at the cell surface, it is predominantly localized to the Golgi compartment . Recent research has shown BCMA's involvement in multiple myeloma pathogenesis and breast cancer progression, making it an important target for immunotherapeutic approaches .

What are the characteristic expression patterns of TNFRSF17/BCMA?

TNFRSF17/BCMA expression has been documented in:

• Immune organs and mature B cell lineages

• Multiple myeloma cells, including the RPMI8226 human myeloma cell line

• Certain breast cancer cells where it mediates cancer progression

Western blot analysis typically reveals BCMA at molecular weights of approximately 18 kDa and 23 kDa, likely representing different glycosylation states or isoforms of the protein . Flow cytometry can effectively detect cell surface expression, as demonstrated in studies with RPMI8226 human myeloma cells .

What are the primary research applications for TNFRSF17/BCMA antibodies?

Based on available data, key research applications include:

These applications enable researchers to investigate BCMA expression, protein interactions, signaling pathways, and potential as a therapeutic target across multiple disease models.

How can researchers effectively evaluate TNFRSF17/BCMA antibody specificity?

A robust validation strategy for TNFRSF17/BCMA antibodies should include:

• Genetic validation using BCMA knockdown/knockout models. This can be accomplished using siRNA targeting BCMA, as demonstrated in studies where T47D cells were transfected with siRNA for BCMA using Lipofectamine 2000 .

• Multiple antibody validation by comparing results with antibodies targeting different BCMA epitopes.

• Recombinant expression testing against cells overexpressing BCMA compared to vector controls.

• Cross-reactivity assessment with related TNF receptor family members, particularly TACI which shares high homology with BCMA.

• Application-specific controls including isotype controls for flow cytometry (as shown in studies with RPMI8226 cells where isotype control antibody was used alongside anti-BCMA antibody) .

• Positive controls using cell lines with documented BCMA expression such as RPMI8226 human myeloma cells .

What protocols optimize TNFRSF17/BCMA detection in Western blotting?

For optimal Western blot detection of TNFRSF17/BCMA:

• Sample preparation: Use lysis buffers that effectively extract membrane proteins while preserving epitope integrity. Consider that BCMA localizes predominantly to the Golgi, requiring thorough extraction protocols.

• Gel electrophoresis: Optimize resolution in the 18-23 kDa range where BCMA typically appears.

• Transfer conditions: Use PVDF membranes and optimize transfer conditions for small proteins.

• Blocking: 5% non-fat dry milk or BSA in TBST typically provides good results.

• Primary antibody: Incubate with anti-BCMA antibody at 1:1000 dilution (for CST antibody #47988) , typically overnight at 4°C.

• Washing: Perform thorough washing with TBST to minimize background.

• Secondary antibody: Use an appropriate HRP-conjugated secondary antibody.

• Controls: Include positive control (BCMA-expressing cells) and negative control (BCMA-negative or knockdown cells).

• Expected results: Anticipate bands at approximately 18 kDa and 23 kDa representing different forms of BCMA .

How should researchers investigate the differential roles of membrane-bound versus soluble BCMA?

This is a critical consideration as "soluble BCMA and BCMA released in vesicles impacts on CAR-BCMA activity" . To effectively distinguish between membrane-bound and soluble forms:

• Selective isolation strategies:

  • Use ultracentrifugation to separate vesicle-associated BCMA

  • Employ cell fractionation to isolate membrane-bound BCMA

  • Use immunoprecipitation from culture supernatants to capture soluble BCMA

• Antibody selection considerations:

  • Choose antibodies that recognize epitopes retained in soluble forms

  • Consider using antibody pairs that can distinguish between forms

• Experimental approaches:

  • Compare BCMA detection in cell lysates versus culture supernatants

  • Evaluate the impact of protease inhibitors on BCMA shedding

  • Use siRNA knockdown to confirm specificity of detected forms

• Functional analysis:

  • Assess the impact of soluble BCMA on CAR-T cell efficacy

  • Determine whether soluble BCMA retains signaling capability

  • Investigate whether soluble BCMA acts as a decoy receptor

How can TNFRSF17/BCMA antibodies be utilized to study BCMA-mediated signaling pathways?

BCMA signaling primarily occurs through the JNK pathway and potentially involves NFκB activation . To investigate these pathways:

• Signaling cascade analysis:

  • Use anti-BCMA antibodies in combination with phospho-specific antibodies against JNK to monitor activation following BCMA engagement

  • Employ luciferase reporter assays with NFκB response elements to assess transcriptional activation (as demonstrated in studies where cells were transfected with pNFκB-Luc plasmid and treated with BAFF or APRIL)

• Inhibitor studies:

  • Compare BCMA-dependent responses in the presence of specific JNK inhibitors such as SP600125 (10 μM)

  • Use complementary approaches with shRNA against JNK1 or JNK2 to confirm pathway specificity

• Co-immunoprecipitation:

  • Use anti-BCMA antibodies (dilution 1:50 for immunoprecipitation) to pull down signaling complexes

  • Identify interaction partners through Western blotting or mass spectrometry

• Temporal analysis:

  • Examine time-dependent changes in BCMA localization and downstream signaling following ligand binding

What methodologies effectively measure TNFRSF17/BCMA-ligand interactions?

To characterize interactions between BCMA and its ligands (APRIL and BAFF):

• ELISA-based interaction assays:

  • Use anti-BCMA antibodies as capture antibodies in sandwich ELISA formats

  • Measure binding of recombinant APRIL or BAFF

• Flow cytometry approaches:

  • Detect BCMA on cells such as RPMI8226 human myeloma cells using validated antibodies

  • Assess competitive binding of labeled ligands

• Functional readouts:

  • Measure downstream effects such as increased mammosphere formation following APRIL or BAFF treatment (100 ng/ml) which increases mammosphere numbers by approximately 40% after 9 days

  • Quantify changes in expression of pluripotency genes (ALDH1A1, KLF4, NANOG) as indicators of BCMA signaling

• Inhibition studies:

  • Use antibodies that block specific BCMA epitopes to prevent ligand binding

  • Compare effects of APRIL and BAFF individually to determine ligand-specific outcomes

How can researchers employ TNFRSF17/BCMA antibodies to evaluate CAR-T cell therapies?

BCMA has emerged as a promising target for CAR-T cell therapy, particularly in multiple myeloma. Researchers have developed both murine (ARI2m) and humanized (ARI2h) CAR-T cells targeting BCMA, with the humanized version showing comparable efficacy but lower toxicity profile . To evaluate such therapies:

• Target expression analysis:

  • Use flow cytometry with anti-BCMA antibodies to quantify expression levels on target cells

  • Assess heterogeneity of BCMA expression within patient samples

• Comparative binding studies:

  • Compare binding properties of therapeutic CAR constructs versus research antibodies

  • Identify potential epitope overlap or competition

• Monitoring studies:

  • Track changes in BCMA expression during disease progression and following therapy

  • Measure soluble BCMA levels, which can impact CAR-BCMA activity

• Efficacy predictors:

  • Correlate pre-treatment BCMA expression levels with clinical responses to CAR-T therapy

  • Investigate resistance mechanisms related to BCMA modulation

What experimental approaches illuminate TNFRSF17/BCMA's role in cancer stemness?

Research has revealed that BCMA signaling increases cancer stem cell properties in breast cancer models . To investigate this phenomenon:

• Stemness marker correlation:

  • Examine co-expression of BCMA with stemness markers (ALDH1A1, KLF4, NANOG)

  • Quantify changes in these markers following APRIL or BAFF treatment (100 ng/ml)

• Functional assays:

  • Assess mammosphere formation capacity after BCMA activation, which increases mammosphere numbers by approximately 40% after 9 days of incubation

  • Compare anchorage-independent growth capabilities

• Signaling pathway analysis:

  • Investigate the role of JNK signaling using specific inhibitors like SP600125 (10 μM)

  • Evaluate transcription factor activation using reporter assays

• EMT connection:

  • Measure vimentin/keratin expression ratios following BCMA activation, as APRIL and BAFF increase epithelial-to-mesenchymal transition

  • Quantify migratory capacity changes in response to APRIL and BAFF treatment (100 ng/ml)

Why do researchers observe multiple molecular weight bands for TNFRSF17/BCMA in Western blots?

Western blot analysis typically reveals BCMA at molecular weights of approximately 18 kDa and 23 kDa . This pattern may result from:

• Post-translational modifications:

  • Different glycosylation states of BCMA

  • Phosphorylation or other modifications affecting mobility

• Protein variants:

  • Alternative splicing generating different BCMA isoforms

  • Proteolytic processing yielding truncated forms

• Technical considerations:

  • Sample preparation methods affecting protein integrity

  • Incomplete denaturation causing aberrant migration

• Biological factors:

  • Membrane-bound versus soluble BCMA forms

  • Vesicle-associated BCMA with altered properties

To differentiate between these possibilities, researchers should perform validation experiments including:

• Deglycosylation treatments

• BCMA knockdown verification of band specificity

• Comparison of different sample preparation methods

What factors can interfere with TNFRSF17/BCMA antibody binding in experimental systems?

Several factors may compromise TNFRSF17/BCMA antibody binding:

• Soluble BCMA interference:

  • Soluble BCMA and vesicle-released BCMA can compete for antibody binding

  • Pre-clearing samples may be necessary in certain applications

• Epitope accessibility issues:

  • BCMA's predominant localization in the Golgi compartment may require appropriate permeabilization protocols

  • Binding of APRIL or BAFF (100 ng/ml concentrations typically used in research) may mask certain epitopes

• Technical considerations:

  • Fixation methods can alter epitope conformation

  • Buffer conditions may affect antibody-antigen interactions

• Biological variables:

  • Expression levels vary across cell types and disease states

  • Androgen treatment can increase APRIL transcription, potentially affecting BCMA-APRIL interactions

What emerging applications for TNFRSF17/BCMA antibodies should researchers consider?

Emerging research applications for TNFRSF17/BCMA antibodies include:

• Multiparameter analysis:

  • Combining BCMA detection with stemness markers to identify cancer stem cell populations

  • Integrating BCMA status with treatment response markers

• Therapeutic development:

  • Monitoring BCMA expression during novel immunotherapy development

  • Developing antibody-drug conjugates targeting BCMA

• Microenvironment studies:

  • Investigating BCMA's role in tumor-immune interactions

  • Exploring paracrine signaling between BCMA-expressing cells and the microenvironment

• Resistance mechanism investigation:

  • Using BCMA antibodies to track antigen loss or modulation during therapy

  • Studying BCMA's role in therapy resistance through EMT and stemness induction