PRPF40B Antibody

What is PRPF40B and why is it significant in research?

PRPF40B (PRP40 pre-mRNA processing factor 40 homolog B) is the human ortholog of the essential yeast splicing factor Prp40, involved in pre-mRNA splicing. It plays a role in 5' splice site recognition and functions primarily as a splicing repressor, inducing a net increase in exon inclusion when knocked out . PRPF40B is significant in research due to its:

• Role in regulating hundreds of alternative splicing targets, particularly those with weak splice sites and A-rich downstream intronic motifs

• Involvement in repressing hypoxia in myeloid cells

• Potential contribution to leukemogenesis when its expression is reduced

• Mutations found in myelodysplastic syndrome (MDS) patients

• Influence on apoptotic gene expression and cell survival

What are the recommended applications for PRPF40B antibodies?

Based on validated commercial antibodies, PRPF40B antibodies have been successfully employed in multiple applications with specific recommended dilutions:

It's recommended to titrate the antibody in each specific testing system to obtain optimal results .

What is the molecular weight of PRPF40B that should be detected by antibodies?

When using PRPF40B antibodies in Western blot applications, researchers should be aware of potential differences between calculated and observed molecular weights:

• Calculated molecular weight: 99 kDa (871 amino acids)

• Observed molecular weight: 130-150 kDa

This discrepancy may be due to post-translational modifications or the specific protein conformation of PRPF40B.

How can I validate PRPF40B knockout models using antibodies?

For validating PRPF40B knockout models, a comprehensive approach combining DNA, RNA, and protein detection is recommended:

• DNA level: PCR genotyping to confirm deletion of target exons (e.g., exons 5-8 as done in Lorenzini et al.)

• RNA level:

  • Perform qRT-PCR using primers targeting multiple exon regions:

    • Primers spanning deleted region (should show complete absence)

    • Primers targeting regions upstream and downstream of deletion (may show reduced expression due to nonsense-mediated decay)

• Protein level:

  • Western blot analysis using antibodies targeting epitopes outside the deleted region

  • Consider antibody epitope location when interpreting results (e.g., C-terminal antibodies may not detect N-terminal fragments)

Research by Lorenzini et al. demonstrated that PRPF40B knockout validation should include assessment of potential truncated proteins, as their CRISPR/Cas9 deletion generated an mRNA potentially encoding a 77 amino acid (8 kDa) peptide that couldn't be detected by their C-terminal antibody .

What controls should be included when using PRPF40B antibodies in immunofluorescence studies?

When conducting immunofluorescence studies with PRPF40B antibodies, include these essential controls:

• Positive controls:

  • Cell lines with validated PRPF40B expression: Y79 cells, K-562 cells

  • Tissue samples: Mouse testis tissue

• Negative controls:

  • PRPF40B knockout cells (if available)

  • Primary antibody omission

  • Isotype control (rabbit IgG)

• Colocalization controls:

  • Phosphorylated SRSF2 (SC35) antibody to mark nuclear speckles, as PRPF40B shows significant overlap with these structures

  • Additional splicing factor markers (SF1, U2AF65) that directly interact with PRPF40B

• Expression validation:

  • EGFP-tagged PRPF40B can confirm the nuclear speckle localization pattern

How can I differentiate between PRPF40B and its paralog PRPF40A in my experiments?

Distinguishing between PRPF40B and its paralog PRPF40A presents a significant challenge due to their high homology (51-54% identity and 65-68% similarity) . To effectively differentiate between these paralogs:

• Antibody selection:

  • Use antibodies with confirmed specificity, targeting less conserved regions

  • Validate antibody specificity using overexpression and knockout controls

  • Consider using epitope-tagged constructs when studying overexpression

• Expression pattern analysis:

  • PRPF40A is highly expressed in AML and blood cancers

  • PRPF40B shows low expression in AML compared to solid tumors

  • Examine tissue-specific expression: PRPF40B has lowest expression in neutrophils/granulocytes among myeloid cells

• Functional discrimination:

  • PRPF40A knockdown affects cell viability and proliferation

  • PRPF40B knockdown has minimal effect on these parameters

  • PRPF40A and PRPF40B show opposite correlations with HIF1A expression

• Western blot mobility:

  • Note that PRPF40A has slower mobility compared to PRPF40B

How should I design rescue experiments for PRPF40B loss-of-function studies?

Based on the Lorenzini et al. study, effective rescue experiments for PRPF40B loss-of-function should follow these methodological steps:

• Expression vector selection:

  • Use PRPF40B isoform C for rescue expressions

  • Consider expression optimization to match endogenous levels

• Transfection optimization:

  • Titrate plasmid amounts (e.g., 100 ng of PRPF40B plasmids) to achieve expression comparable to endogenous levels

  • Allow sufficient expression time (72 hours post-transfection showed optimal results)

• Experimental conditions:

  • Include these experimental groups:

    • Parental cells (positive control)

    • Knockout cells (negative control)

    • Knockout cells + wild-type PRPF40B (rescue control)

    • Knockout cells + mutant PRPF40B (test condition)

    • For modeling heterozygous mutations: Knockout cells + 1:1 mixture of wild-type and mutant PRPF40B

• Validation metrics:

  • RNA and protein expression levels comparable to parental cells

  • Functional readouts (splicing patterns, phenotypic rescue)

  • Minimum of three independent replicates

Why might I observe multiple bands when using PRPF40B antibodies in Western blot?

Multiple bands observed in Western blot with PRPF40B antibodies may occur for several reasons:

• Cross-reactivity with PRPF40A: Due to high homology between PRPF40B and PRPF40A (51-54% identity), antibodies may recognize both proteins

• Alternative isoforms: PRPF40B has multiple isoforms (e.g., isoform C was used in Lorenzini's rescue experiments)

• Post-translational modifications: The discrepancy between calculated (99 kDa) and observed (130-150 kDa) molecular weights suggests extensive modifications

• Degradation products: Especially in protocols with insufficient protease inhibition

• Cell-type specific expression patterns: Different cell lines may express different isoforms or modification patterns, as shown in Western blot data:

  Cell/Tissue Type	Band Pattern
  RT-4 cell lysate	Multiple bands
  U-251 MG cell lysate	Multiple bands
  Human liver	Different pattern
  Human tonsil	Different pattern
  Y79 cells	Positive control
  K-562 cells	Positive control
  Mouse testis	Positive control

To minimize these issues, use positive controls, knockout validation, and antibodies targeting specific epitopes.

What are the optimal fixation and permeabilization methods for PRPF40B immunostaining?

For successful immunostaining of PRPF40B in nuclear speckles:

• Fixation options:

  • Paraformaldehyde (4%) for 10-15 minutes at room temperature preserves nuclear architecture while maintaining epitope accessibility

  • Methanol fixation (-20°C) for 10 minutes may better preserve nuclear proteins

• Permeabilization methods:

  • 0.1-0.5% Triton X-100 in PBS for 5-10 minutes for paraformaldehyde-fixed cells

  • No additional permeabilization needed for methanol-fixed cells

• Blocking conditions:

  • 5% normal serum (matched to secondary antibody host) with 1% BSA in PBS for 30-60 minutes

• Antibody incubation:

  • Primary: overnight at 4°C at optimized dilution in blocking buffer

  • Secondary: 1-2 hours at room temperature

• Nuclear counterstain:

  • DAPI or Hoechst for nuclear visualization

  • Consider the emission spectrum when designing co-staining experiments with SRSF2 (SC35)

How does PRPF40B expression correlate with cancer progression, particularly in myeloid malignancies?

PRPF40B shows distinct expression patterns in myeloid malignancies with potential prognostic significance:

• Expression in Acute Myeloid Leukemia (AML):

  • PRPF40B displays consistently low expression in AML compared to solid tumors

  • In contrast, its paralog PRPF40A shows high expression in AML

• Correlation with hypoxia regulators:

  • PRPF40B negatively correlates with HIF1A expression

  • PRPF40A positively correlates with HIF1A expression

  • This opposing correlation is notable given the heterogeneity of AML samples

• Role in Myelodysplastic Syndrome (MDS):

  • PRPF40B mutations (missense) have been detected in MDS patients

  • These mutations (e.g., P383L and P540S) may result in loss of function

• Functional implications:

  • PRPF40B appears to repress hypoxia in myeloid cells

  • Low PRPF40B expression might contribute to leukemogenesis

  • PRPF40B depletion increases Fas/CD95 receptor number and cell apoptosis

These findings suggest that PRPF40B antibodies could be valuable tools for investigating the role of this splicing factor in cancer progression and potentially as a diagnostic or prognostic marker.

What splicing events are regulated by PRPF40B and how can antibodies help investigate these mechanisms?

PRPF40B regulates specific splicing events with the following characteristics:

• Splicing target profile:

  • Primarily acts as a splicing repressor (knockout leads to increased exon inclusion)

  • Affects exons with A-rich downstream intronic motifs

  • Regulates exons with weak splice sites, especially 5' splice sites

  • Influences cotranscriptional splicing events

• Pathway regulation:

  • Loss of PRPF40B induces KLF1 transcriptional signature

  • Regulates genes involved in iron metabolism, hypoxia

  • Affects cholesterol biosynthesis and Akt/MAPK signaling pathways

• Specific target genes:

  • Fas/CD95 alternative splicing (weak 5' and 3' splice sites and exonic sequences are required for PRPF40B function)

  • Cytokine/chemokine genes that are HIF1A targets

• Antibody-based investigation methods:

  • Chromatin immunoprecipitation (ChIP) to study cotranscriptional splicing

  • RNA immunoprecipitation (RIP) to identify direct RNA targets

  • Immunoprecipitation followed by mass spectrometry to identify protein interaction partners

  • Proximity ligation assays to confirm interactions with other splicing factors (SF1, U2AF65)

• Complementary approaches:

  • RNA-seq after PRPF40B knockout/knockdown to identify global splicing changes

  • RT-PCR validation of specific splicing events

  • Immunofluorescence colocalization with splicing machinery components

How do PRPF40B antibodies perform across different myeloid cell lines and patient samples?

Research data indicates varied performance of PRPF40B antibodies across myeloid cell models:

• Cell line performance:

  Cell Line	Applications	Notes
  K-562	WB, IF	Validated positive control , used in knockout studies
  HL-60	WB	Used in knockdown studies for PRPF40A/B comparison
  Y79	WB, IP	Strong signal in validation studies
  U-251 MG	WB	Validated in commercial antibody testing

• Patient sample considerations:

  • PRPF40B has low expression in AML compared to solid tumors

  • Detection in patient samples requires sensitive antibodies

  • Consider enrichment techniques before immunodetection

• Tissue specificity:

  • Strong signal in mouse testis tissue

  • Detectable in human cerebellum using IHC-P methods

  • Variable detection in human plasma, liver, and tonsil samples

• Technical recommendations:

  • Use fresh samples when possible

  • For archival samples, optimize antigen retrieval methods

  • Include positive controls (K-562 cells) in all experiments

How do the phenotypic effects of PRPF40A and PRPF40B differ in experimental models, and how can antibodies help distinguish these effects?

PRPF40A and PRPF40B show distinct phenotypic effects that can be distinguished with appropriate antibody-based approaches:

• Cell viability and proliferation:

  • PRPF40A knockdown significantly reduces cell viability and increases cell death

  • PRPF40B knockdown has minimal effect on viability

  • Interestingly, PRPF40B overexpression can partially rescue cell death caused by PRPF40A knockdown

• Cell cycle effects:

  • PRPF40A-KD cells show reduced proliferation with G1 phase accumulation

  • This proliferation phenotype is not rescued by PRPF40B overexpression

• Differentiation markers:

  • PRPF40A knockdown upregulates CD86 (T-cell costimulatory molecule)

  • PRPF40A-KD induces only slight (~10%) increase in CD11b, an early myeloid differentiation marker

• Antibody-based discrimination methods:

  • Flow cytometry with specific antibodies to measure differentiation markers (CD11b, CD14, CD86)

  • Immunofluorescence to assess subcellular localization differences

  • Western blot analysis noting the different observed molecular weights

  • Co-immunoprecipitation to identify distinct protein interaction partners

• Transcriptomic validation:

  • RNA-seq after specific knockdown to identify distinct splicing targets

  • RT-PCR validation of paralog-specific splicing events

These distinct phenotypic effects highlight the non-redundant functions of PRPF40A and PRPF40B, despite their sequence similarity, and demonstrate the importance of specific antibodies in delineating their roles.

What emerging applications for PRPF40B antibodies should researchers consider?

Several emerging applications for PRPF40B antibodies show promise for advancing our understanding of splicing regulation:

• Single-cell protein analysis:

  • Integration with mass cytometry for single-cell protein expression profiling

  • Correlation of PRPF40B levels with differentiation states in heterogeneous populations

• Spatial transcriptomics integration:

  • Combining immunofluorescence with spatial transcriptomics to correlate PRPF40B localization with splicing outcomes in tissue context

• Therapeutic target validation:

  • Using antibodies to validate PRPF40B as a potential therapeutic target in hypoxia-dependent malignancies

  • Development of antibody-drug conjugates for targeting PRPF40B-expressing cells

• Diagnostic applications:

  • Assessment of PRPF40B/PRPF40A expression ratio as a diagnostic or prognostic marker in myeloid malignancies

  • Immunohistochemical evaluation of PRPF40B in bone marrow biopsies

• Functional proteomics:

  • Proximity labeling approaches (BioID, APEX) using PRPF40B-fusion proteins to identify the complete interactome

  • Mapping dynamic changes in PRPF40B interactions during differentiation or stress response

These applications could significantly expand our understanding of PRPF40B's role in normal and malignant hematopoiesis.

How might PRPF40B missense mutations found in MDS patients affect antibody epitope recognition?

The impact of PRPF40B missense mutations on antibody recognition requires careful consideration:

• Documented mutations in MDS:

  • P383L and P540S mutations have been identified in MDS patients

  • Additional missense mutations occur throughout the open reading frame

• Epitope considerations:

  • Commercial antibodies target different regions:

    • Proteintech 16929-1-AP: PRPF40B fusion protein Ag10361

    • Abcam ab122474: Recombinant fragment within Human PRPF40B aa 750-850

  • Mutations may alter protein conformation and epitope accessibility

• Testing recommendations:

  • Validate antibody recognition of mutant PRPF40B proteins using overexpression systems

  • Consider using multiple antibodies targeting different epitopes

  • Include wild-type controls alongside mutant samples

• Functional considerations:

  • Research suggests MDS mutations may cause loss of function

  • Antibody-based methods can help determine if mutations affect:

    • Protein stability and turnover

    • Nuclear localization and speckle association

    • Interaction with binding partners (SF1, U2AF65)