tssc4 Antibody

What is TSSC4 and what functional domains should my antibody target?

TSSC4 is a tumor suppressor protein located in a chromosomal region containing multiple tumor suppressor genes including CDKN1C/p57/KIP2, PHLDA2/TSSC3, and H19 . It functions as:

• A component of U5 snRNP essential for RNA splicing

• An autophagy inhibitor that interacts with LC3-I via a conserved LC3-interacting region (LIR) motif

• A regulator of alternative splicing affecting oncogene expression

When selecting antibodies, consider those targeting:

• The conserved LIR motif (residues 97-100, specifically the FDCL sequence) if studying autophagy interactions

• The TSSC4 domains critical for interaction with U5 snRNP and PRPF19 complex if investigating splicing mechanisms

How should I design experiments to study TSSC4's role in autophagy using antibodies?

To investigate TSSC4's role in autophagy inhibition:

• Baseline comparison: Compare LC3B-II levels in control vs. TSSC4-knockdown/knockout cells by Western blot. TSSC4 knockout typically increases autophagy, which can be measured by increased LC3B-II levels in the presence of chloroquine (CQ) .

• Interaction studies: Co-immunoprecipitation experiments using anti-TSSC4 antibodies to pull down LC3-I. The FDCL motif (residues 97-100) is critical for this interaction .

• Functional validation: Compare TSSC4 wild-type vs. TSSC4M (F97A, L100A mutant) expression to confirm the functional importance of the LIR motif. TSSC4 inhibits basal autophagy and TMZ-induced autophagy through its conserved LIR, while TSSC4M does not affect autophagy levels .

• Cell death assays: Measure TMZ-induced autophagy-dependent cell death in the presence of TSSC4 or TSSC4M. TSSC4 inhibits TMZ-induced cell death, unlike TSSC4M .

What controls are essential when using TSSC4 antibodies in cancer research models?

When studying TSSC4 in cancer models, include:

• Knockout/knockdown validation: Confirm antibody specificity by including TSSC4-knockout cells as negative controls. Published studies have used CRISPR/Cas9 to generate TSSC4-knockout cell lines in HeLa, A549, and U251 cells .

• Expression validation: When overexpressing wild-type or mutant TSSC4, confirm expression levels by Western blot using anti-TSSC4 or anti-tag antibodies if using tagged versions .

• Functional controls: Include the TSSC4M mutant (F97A, L100A) as a control when studying autophagy, as it lacks the ability to inhibit autophagy but maintains other functions .

• Autophagy pathway controls: When studying TSSC4's role in autophagy, include autophagy inhibitors (3-MA) or ATG7 knockdown as pathway controls .

How can I optimize co-immunoprecipitation assays to study TSSC4 interactions with splicing factors?

TSSC4 interacts with multiple proteins in the U5 snRNP complex. For successful co-IP studies:

• Antibody selection: Use antibodies targeting different epitopes of TSSC4 to avoid interference with protein interaction sites. Alternatively, use tag-based approaches (HA-tagged or GFP-tagged TSSC4) for cleaner results .

• Nuclear extraction optimization: Since TSSC4 functions primarily in the nucleus with the U5 snRNP complex, optimize nuclear extraction protocols to maintain protein-protein interactions. TSSC4 associates with U5 snRNP proteins (PRPF8, EFTUD2, SNRNP200) .

• Cross-validation approach: Confirm interactions by both forward and reverse co-IP (pulling down with anti-TSSC4 antibody and with antibodies against suspected interaction partners) .

• Quantitative analysis: Employ SILAC (Stable Isotope Labeling with Amino acids in Cell culture) combined with immunoprecipitation for unbiased quantification of interaction partners, as demonstrated in previous studies .

What are the methodological considerations for investigating TSSC4's effect on alternative splicing?

To study TSSC4's role in splicing regulation:

• RNA-seq experimental design: Compare transcriptomes of TSSC4-knockout or knockdown cells with controls to identify alternative splicing events. Previous studies revealed widespread alterations in splicing patterns, particularly intron retention events .

• Validation strategy: Use RT-qPCR to validate identified alternative splicing events and changes in oncogene expression (e.g., VEGFC, JAG1, BCL2L1, IRS1) .

• Protein interaction analysis: Use anti-TSSC4 antibodies to investigate interactions with U5 snRNP components that may explain splicing dysregulation. KEGG pathway analysis of TSSC4-regulated AS changes showed enrichment of the mRNA surveillance pathway and spliceosome .

• Functional verification: Correlate splicing changes with phenotypic outcomes such as cell proliferation or tumor formation. TSSC4 deficiency leads to aberrant alternative splicing and increased expression of oncogenes, supporting its role as a tumor suppressor .

How can I troubleshoot inconsistent TSSC4 detection in Western blots?

If experiencing difficulties detecting TSSC4:

• Sample preparation optimization:

  • TSSC4 shuttles between cytoplasm and nucleus; therefore, prepare whole cell lysates rather than only cytoplasmic fractions

  • Use RIPA buffer with protease inhibitors to prevent degradation

• Loading control selection:

  • Most published studies use β-actin (ACTB) as a loading control for TSSC4 Western blots

  • Ensure consistent loading (10-20 μg total protein per lane)

• Antibody optimization:

  • Test different dilutions within the recommended range (1:500-1:2000)

  • Consider longer primary antibody incubation (overnight at 4°C)

  • Expected molecular weight is approximately 34 kDa

• Enhanced detection methods:

  • For low abundance, use enhanced chemiluminescence substrates or consider fluorescent secondary antibodies

How do I design experiments to study TSSC4's dual role in autophagy and splicing?

To investigate the connection between TSSC4's functions:

• Domain-specific mutants: Create targeted mutations in different functional domains:

  • TSSC4M (F97A, L100A) for autophagy function

  • Mutations in domains critical for U5 snRNP interaction for splicing function

• Sequential experimental design:

  • First establish effects on autophagy using LC3B-II Western blots with and without chloroquine

  • Then examine splicing alterations using RNA-seq or RT-PCR analysis of alternative splicing events

  • Correlate both functions with cancer cell phenotypes (proliferation, tumorsphere formation)

• Cross-pathway analysis:

  • Determine if splicing dysregulation affects autophagy genes

  • Examine if autophagy inhibition affects expression or localization of splicing factors

• Time-course studies:

  • Monitor TSSC4 activity in both pathways following drug treatment, as TSSC4 contributes to drug resistance by inhibiting TMZ-induced cell death in cancer cells

How can TSSC4 antibodies be used to investigate its tumor suppressor role in different cancer types?

TSSC4 knockdown increases cell growth in multiple cancer cell lines (MDA-MB-231, U87, U373, HeLa) . To investigate its tumor suppressor mechanisms:

• Tissue microarray analysis:

  • Use anti-TSSC4 antibodies (1:20-1:200 dilution) for IHC to compare TSSC4 expression across different cancer types and stages

  • Correlate expression with clinical outcomes and other tumor suppressor markers

• Mechanism-specific experimental designs:

  • For autophagy inhibition: Compare tumorsphere formation in cells expressing TSSC4 vs. TSSC4M to determine if autophagy inhibition is responsible for reduced tumorsphere formation (up to 44% reduction has been observed)

  • For splicing regulation: Examine if TSSC4 knockdown causes consistent patterns of oncogene upregulation across cancer types

• Combination drug studies:

  • Use TSSC4 antibodies to monitor protein expression and interactions when treating cells with TMZ and autophagy inhibitors

  • TSSC4 knockout increases TMZ-induced cell death while autophagy inhibitor 3-MA inhibits TMZ-induced cell death by at least 30%

• In vivo models:

  • Develop xenograft models with TSSC4-knockout or TSSC4-overexpressing cancer cells

  • Use antibodies to confirm TSSC4 status in tumors and analyze correlation with growth rates

By applying these methodologies with appropriate controls and validation steps, researchers can effectively use TSSC4 antibodies to advance our understanding of this multifunctional protein in cancer biology.