SF3A2 Antibody

What is SF3A2 and why is it important in cellular research?

SF3A2 (Splicing Factor 3a, Subunit 2, 66kDa) is a crucial component of the spliceosome, specifically within the U2 snRNP complex. It functions in pre-mRNA splicing, a fundamental cellular process for gene expression regulation. Recent studies have shown SF3A2 plays direct roles in mitotic processes, with evidence that depleting SF3A2 disrupts mitotic division within minutes, indicating functions beyond splicing regulation . Additionally, SF3A2 has been implicated in cancer progression, particularly in triple-negative breast cancer (TNBC) where it promotes tumor growth and confers cisplatin resistance . The protein's multifunctional nature makes SF3A2 antibodies valuable tools for investigating both splicing mechanisms and cancer biology.

What are the common applications for SF3A2 antibodies?

SF3A2 antibodies are utilized across multiple experimental applications:

These applications make SF3A2 antibodies versatile tools for examining protein expression, localization, and interaction networks .

How do researchers validate SF3A2 antibody specificity?

Methodological validation of SF3A2 antibodies typically involves multiple complementary approaches:

• RNAi knockdown experiments - Western blotting demonstrating band reduction following SF3A2 siRNA/shRNA treatment is considered strong validation. Studies show effective knockdown reduces the 66 kDa band to at least 20% of control levels .

• Molecular weight confirmation - Verification that the detected band appears at the expected molecular weight (66 kDa for human SF3A2) .

• Immunogen specificity - Many validated antibodies are generated against specific epitopes, such as KLH-conjugated synthetic peptides between amino acids 166-194 from the central region of human SF3A2 .

• Cross-reactivity assessment - Testing against multiple species to confirm predicted reactivity with human, mouse, and rat samples .

• Orthogonal detection methods - Combining multiple applications (WB, IHC, IF) to confirm consistent target recognition .

How have SF3A2 antibodies contributed to understanding branch site recognition in splicing mechanisms?

SF3A2 antibodies have provided crucial insights into spliceosome assembly mechanics, particularly regarding branch site (BS) recognition. In a landmark study examining cancer-associated SF3B1 mutations, researchers used SF3A2 antibodies to isolate U2 snRNP complexes from chromatin-bound fractions, enabling identification of protected branch point sequences across the transcriptome .

The methodology involved:

• Immunopurification of U2 complexes using anti-SF3A2 antibodies targeting the C-terminal peptide (MLRPPLPSEGPGNIP)

• Isolation of RNA from high-molecular-weight extracted complexes

• Removal of U2 snRNA by antisense oligonucleotide-directed RNase H degradation

• Sequencing of remaining protected RNA fragments

This approach demonstrated that SF3A2 antibodies can detect allele-specific branch points bound by endogenous U2 snRNP, revealing that the SF3B1 K700E mutation causes widespread alterations in branch site recognition beyond previously recognized patterns . The technique is particularly valuable because it works effectively in heterozygous cell systems without requiring epitope tagging of U2 components, making it applicable for patient-derived samples.

What role does SF3A2 play in cancer progression and how can antibodies help elucidate these mechanisms?

Recent research has identified SF3A2 as a potential oncogenic factor and therapeutic target, particularly in triple-negative breast cancer (TNBC). SF3A2 antibodies have been instrumental in demonstrating that:

• SF3A2 is aberrantly upregulated in TNBC tissues compared to adjacent normal tissues, correlating with poor prognosis .

• SF3A2 accelerates TNBC progression by regulating alternative splicing of the makorin ring finger protein 1 (MKRN1) gene, promoting expression of the oncogenic MKRN1-T1 isoform .

• SF3A2 contributes to cisplatin resistance through multiple mechanisms:

  • Regulation of extrinsic apoptosis via the MKRN1-FADD pathway

  • Modulation of intrinsic apoptosis through DNA damage response pathways

  • Positive regulation of UBR5 protein levels, which is consistent with previously reported oncogenic activities in TNBC progression

Methodologically, researchers have employed SF3A2 antibodies for:

• Western blotting to monitor SF3A2 expression levels and correlation with chemoresistance

• Immunoprecipitation to identify novel protein interactions, including UBR5 as a binding partner

• Immunofluorescence to demonstrate nuclear co-localization with interaction partners

This research highlights how SF3A2 antibodies can be applied beyond basic splicing research to investigate cancer pathogenesis and potential therapeutic vulnerabilities.

How are SF3A2 antibodies utilized in studying protein-protein interactions within the spliceosome?

SF3A2 antibodies have proven valuable for investigating the complex protein-protein interaction network within the spliceosome. Methodological approaches include:

• Co-immunoprecipitation (Co-IP) assays: SF3A2 antibodies have been used to purify spliceosomal complexes, revealing interactions with:

  • Other SF3A subunits (SF3A1, SF3A3)

  • SF3B complex components (SF3B1, SF3B2, SF3B3)

  • U2 snRNP-associated proteins (DHX15, RBM17)

  • RBM5 and RBM10 (G-patch motif-containing proteins)

• Chromatin-associated complex isolation: SF3A2 antibodies can differentiate between:

  • Nucleoplasmic U2 complexes (containing either SF3A or SF3B subunits, but not both)

  • Chromatin-bound complexes (containing both SF3A and SF3B subunits)

• Comparative proteomics: IP with SF3A2 antibodies followed by mass spectrometry has identified differences in protein composition between wild-type and mutant SF3B1-containing U2 complexes .

This methodological approach has revealed that while SF3A2 associates with multiple spliceosomal proteins, the SF3B1 K700E mutation does not drastically alter core U2 protein composition but rather affects RNA binding specificity.

What are the optimal conditions for using SF3A2 antibodies in immunohistochemistry?

Based on published protocols and manufacturer recommendations, optimal conditions for SF3A2 antibody use in immunohistochemistry include:

It is crucial to include appropriate negative controls (omitting primary antibody) and positive controls (tissues known to express SF3A2) in each experiment. Researchers should also be aware that different fixation methods may affect epitope accessibility, potentially requiring optimization of antigen retrieval conditions .

What specific considerations apply when using SF3A2 antibodies for studying splicing mechanisms?

When investigating splicing mechanisms using SF3A2 antibodies, researchers should consider several methodological factors:

• Nuclear extraction protocols: Since SF3A2 functions primarily in the nucleus as part of the spliceosome, proper nuclear extraction is critical. Studies have shown that separating the high-molecular-weight nuclear fraction (containing chromatin) from the soluble nucleoplasm provides better resolution of active splicing complexes .

• Cross-linking conditions: For capturing transient RNA-protein interactions within the spliceosome, optimized cross-linking protocols may be required. UV cross-linking or formaldehyde treatment has been employed successfully .

• RNase treatment considerations: When isolating protein complexes, controlled RNase treatment can help distinguish between direct protein-protein interactions and RNA-mediated associations. In one study, chromatin-bound complexes were extracted with a combination of DNase and RNase to isolate U2 particles containing SF3A2 .

• Distinguishing isoform-specific effects: When studying alternative splicing regulation by SF3A2, researchers should design experiments that can differentiate between general splicing defects and isoform-specific alterations. For example, RT-PCR assays with isoform-specific primers were used to show that SF3A2 knockdown resulted in decreased MKRN1-T1 isoform levels while increasing MKRN1-T2 isoform levels .

• Minigene reporter assays: To directly assess SF3A2's role in specific splicing events, minigene reporters spanning relevant exons and introns can be constructed. In one study, a minigene spanning MKRN1 exon 5, intron 5, and exon 6 demonstrated that SF3A2 knockdown inhibited inclusion of short exon 5 (from 74% to 17%), while SF3A2 overexpression promoted its inclusion (from 52% to 74%) .

How should researchers design controls when studying SF3A2 using antibody-based approaches?

Effective experimental controls are essential for reliable SF3A2 antibody-based research:

• Knockdown/knockout validation controls:

  • Western blotting following RNAi should demonstrate reduction of the SF3A2 band to at least 20% of control levels

  • Include both negative control siRNA/shRNA and gene-specific targeting constructs

  • For phenotypic studies, rescue experiments with SF3A2 re-expression are crucial to confirm specificity

• Antibody specificity controls:

  • Peptide competition assays using the immunizing peptide (e.g., KLH-conjugated synthetic peptide between 166-194 amino acids)

  • Multiple antibodies targeting different epitopes of SF3A2 to confirm consistent results

  • Testing in cell lines with known SF3A2 expression levels (positive: NIH/3T3 cells, HeLa cells)

• Cross-reactivity controls:

  • When studying SF3A2 in non-human models, validate antibody reactivity in the specific species

  • Test predicted cross-reactivity with mouse, rat, and other species as relevant

• Functional controls:

  • When studying SF3A2's role in splicing, include well-characterized splicing factors as positive controls

  • For U2 snRNP studies, antibodies against other components (SF3A1, SF3B1) can provide complementary data

  • For cell cycle or apoptosis studies, established markers and inhibitors should be included

How can researchers address inconsistent results when using SF3A2 antibodies across different applications?

When confronting inconsistent results with SF3A2 antibodies, researchers should consider these methodological approaches:

• Application-specific optimization:

  • Western blotting: Test multiple blocking agents (BSA vs. milk), transfer conditions, and incubation times

  • Immunohistochemistry: Compare different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Immunofluorescence: Evaluate fixation methods (paraformaldehyde vs. methanol) and permeabilization conditions

• Antibody selection considerations:

  • Different applications may require antibodies recognizing different epitopes

  • Monoclonal antibodies offer consistent results but may have limited epitope recognition

  • Polyclonal antibodies provide broader epitope recognition but potential batch variation

  • For SF3A2, polyclonal antibodies against the central region (AA 166-194) have shown consistent results across applications

• Sample preparation factors:

  • Cell lysis buffers should be optimized for nuclear proteins like SF3A2

  • For chromatin-associated studies, separate high-molecular-weight nuclear fractions from soluble nucleoplasm

  • Consider protein post-translational modifications that might affect antibody recognition

• Cross-validation strategies:

  • Use multiple antibodies targeting different regions of SF3A2

  • Complement antibody-based detection with orthogonal approaches (e.g., mass spectrometry)

  • Include siRNA/shRNA knockdown controls to confirm specificity

What are the key technical challenges when using SF3A2 antibodies for chromatin immunoprecipitation studies?

Chromatin immunoprecipitation with SF3A2 antibodies presents several technical challenges that researchers should address:

• Antibody selection considerations:

  • For SF3A2 ChIP applications, antibodies must recognize native (non-denatured) epitopes

  • The C-terminal peptide of SF3A2 (MLRPPLPSEGPGNIP) has been successfully used for generating ChIP-grade antibodies

  • Antibodies should be validated specifically for ChIP applications before use

• Cross-linking optimization:

  • Standard formaldehyde cross-linking may not efficiently capture transient interactions between SF3A2 and chromatin

  • Combined protein-protein and protein-RNA cross-linking approaches may be needed

  • UV cross-linking can provide higher specificity for direct RNA interactions

• Nuclear extraction challenges:

  • SF3A2 exists in multiple complexes (free vs. spliceosome-bound)

  • Separating chromatin-bound from nucleoplasmic fractions improves signal specificity

  • DNase and controlled RNase treatment helps isolate functional complexes

• Signal-to-noise ratio:

  • Background can result from SF3A2's abundant expression and involvement in multiple complexes

  • Pre-clearing lysates with non-specific IgG is essential

  • Sequential ChIP (re-ChIP) may help isolate specific SF3A2-containing complexes

• Data interpretation considerations:

  • SF3A2 may not directly bind DNA but associates with chromatin through RNA interactions

  • Distinguishing direct from indirect associations requires careful experimental design

  • IP-seq approaches may be more informative than standard ChIP-seq for splicing factors

How can the purity and specificity of SF3A2 antibody immunoprecipitation be improved for proteomic studies?

For proteomic applications requiring high-purity SF3A2 immunoprecipitation, researchers should consider these methodological refinements:

• Antibody purification approaches:

  • Use affinity-purified antibodies that undergo both protein A column purification and peptide affinity purification

  • Consider cross-linking antibodies to beads to prevent antibody contamination in mass spectrometry samples

  • For epitope-tagged SF3A2 studies, anti-Flag antibodies have shown high specificity and efficiency

• Stringency optimization:

  • Adjust salt concentrations in wash buffers to balance complex integrity with background reduction

  • Include non-ionic detergents (0.1% NP-40 or Triton X-100) to reduce non-specific binding

  • Test multiple elution conditions (peptide competition vs. low pH) to identify optimal approach

• Two-step immunoprecipitation strategies:

  • Sequential IP with antibodies against different complex components can increase specificity

  • Tandem affinity purification using tagged SF3A2 provides higher purity

  • For endogenous complexes, combined IP with anti-SF3A2 and anti-SF3B1 antibodies enhances specificity

• Sample fractionation:

  • Pre-fractionation of nuclear extracts (e.g., glycerol gradient or size exclusion chromatography)

  • Separate chromatin-bound from nucleoplasmic fractions before IP

  • This approach revealed that nucleoplasmic and chromatin-bound SF3A2 exist in distinct complexes

• Controls for proteomic analysis:

  • Include IgG control IP from same cellular fractions

  • When studying specific splicing contexts, compare IPs from control and treatment conditions

  • For identifying SF3A2-specific interactors, parallel IP with other spliceosomal proteins helps distinguish common from specific interactions

How can SF3A2 antibodies contribute to understanding cancer-associated splicing factor mutations?

SF3A2 antibodies have emerged as valuable tools for investigating how mutations in splicing factors contribute to cancer development:

• U2 snRNP complex dynamics:

  • SF3A2 antibodies enable purification of intact U2 snRNP complexes from cells harboring cancer-associated mutations

  • This approach revealed that the SF3B1 K700E mutation (common in myelodysplastic syndrome) does not alter core U2 complex composition but affects branch site recognition

• Allele-specific studies:

  • SF3A2 antibodies can isolate complexes containing both wild-type and mutant SF3B1 alleles

  • This allows comparison of how mutations affect splicing mechanisms without requiring epitope tagging

• Branch site profiling:

  • U2 IP-seq using SF3A2 antibodies enables mapping of branch sites bound by U2 across the transcriptome

  • This revealed that SF3B1 K700E causes far more widespread changes in U2/branch site interactions than previously anticipated

• Therapeutic target validation:

  • SF3A2 antibodies have helped establish SF3A2 itself as a potential therapeutic target

  • Research shows SF3A2 promotes triple-negative breast cancer progression and cisplatin resistance

  • Targeting SF3A2 could potentially re-sensitize resistant tumors to standard chemotherapies

What role does SF3A2 play in cisplatin resistance and how can antibody-based approaches help develop new therapeutic strategies?

Recent research using SF3A2 antibodies has uncovered its significant role in cisplatin resistance, particularly in triple-negative breast cancer:

• Mechanisms of cisplatin resistance:

  • SF3A2 regulates both extrinsic and intrinsic apoptosis pathways

  • SF3A2 depletion leads to increased levels of γH2AX (marker for DNA double-strand breaks)

  • SF3A2-knockdown tumors show enhanced sensitivity to cisplatin therapy in vivo

• Methodological approaches:

  • Western blotting with SF3A2 antibodies to monitor expression levels in resistant vs. sensitive cells

  • Immunofluorescence to detect γH2AX foci formation following cisplatin treatment

  • Xenograft models comparing cisplatin response in SF3A2-depleted vs. control tumors

• UBR5-SF3A2-MKRN1 pathway:

  • SF3A2 antibodies helped identify UBR5 as a binding partner and ubiquitin ligase for SF3A2

  • This pathway regulates FADD protein levels and subsequent apoptotic response

  • Targeting this pathway could potentially overcome cisplatin resistance

• Therapeutic implications:

  • SF3A2 inhibition could sensitize resistant tumors to cisplatin

  • Combined targeting of SF3A2 and TRAIL pathways might enhance therapeutic efficacy

  • Biomarker development using SF3A2 antibodies could help identify patients likely to develop resistance

How are SF3A2 antibodies being used to study non-canonical functions beyond splicing regulation?

Beyond its established role in splicing, SF3A2 has emerging non-canonical functions that are being investigated using antibody-based approaches:

• Mitotic regulation:

  • Anti-SF3A2 antibody injections into Drosophila embryos disrupt mitotic division within one minute

  • This rapid effect strongly argues against a splicing-related mechanism

  • Western blotting with SF3A2 antibodies showed that RNAi-mediated depletion affects cell division via the spindle assembly checkpoint (SAC)

• Microtubule association:

  • SF3A2 has been identified as a microtubule-binding and -bundling protein

  • Immunofluorescence with SF3A2 antibodies can visualize its cytoplasmic localization during specific cell cycle phases

  • This non-nuclear function represents a distinct role from its canonical splicing activity

• DNA damage response pathways:

  • SF3A2 depletion results in increased DNA damage (γH2AX foci)

  • SF3A2 antibodies help visualize DNA damage foci formation and co-localization studies

  • This suggests SF3A2 may participate in DNA damage repair independent of its splicing function

• Protein stability regulation:

  • SF3A2 regulates UBR5 protein levels and stability

  • Immunoprecipitation with SF3A2 antibodies followed by ubiquitination assays revealed this bidirectional relationship

  • This establishes SF3A2 as part of a complex regulatory network extending beyond splicing