pspc1 Antibody

What is PSPC1 and what cellular functions does it perform?

PSPC1 (paraspeckle component 1) is a known paraspeckle biomarker and putative transcription factor belonging to the Drosophila behavior/human splicing (DBHS) family . This RNA-binding protein is required for the formation of nuclear paraspeckles and has demonstrated binding affinity for poly(A), poly(G), and poly(U) RNA homopolymers . PSPC1 performs multiple cellular functions including regulation of androgen receptor-mediated gene transcription activity in cooperation with NONO and SFPQ proteins .

Additionally, PSPC1 serves as a regulator of the circadian clock by repressing the transcriptional activator activity of the CLOCK-BMAL1 heterodimer . It also plays a significant role in the regulation of DNA virus-mediated innate immune response by assembling into the HDP-RNP complex, which functions as a platform for IRF3 phosphorylation and subsequent innate immune response activation through the cGAS-STING pathway . Recent research has also identified PSPC1 as a contextual determinant of tumor progression in multiple cancer types, where it can reprogram TGF-β signaling from proapoptotic to prometastatic by hijacking Smad2/3 targeting .

What applications are commercially available PSPC1 antibodies validated for?

Commercial PSPC1 antibodies have been extensively validated for multiple experimental applications. For example, the Proteintech antibody (16714-1-AP) is validated for Western Blot (WB), Immunoprecipitation (IP), Immunofluorescence (IF)/ICC, and ELISA applications . Similarly, the Abcam antibody (ab104238) is suitable for IP, WB, and ICC/IF applications .

The validation data shows positive Western Blot detection in multiple cell lines including HEK-293 cells, HepG2 cells, and PC-3 cells . Immunoprecipitation has been confirmed in HEK-293 cells, while immunofluorescence/ICC has been validated in PC-3 cells . These antibodies demonstrate reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species . For researchers planning experiments, it is important to note that while some species and application combinations have been directly tested and validated, others may be predicted to work based on sequence homology but might require additional optimization .

What are the recommended dilutions and experimental conditions for PSPC1 antibodies?

For optimal experimental results with PSPC1 antibodies, the following dilution ranges are recommended based on application type:

The storage conditions for maintaining antibody activity recommend keeping the antibody at -20°C, where it remains stable for one year after shipment . The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For the 20μl size preparations, it's worth noting that they contain 0.1% BSA . Proper storage and handling are essential for maintaining antibody specificity and sensitivity across multiple experiments.

What is the molecular profile of PSPC1 protein that researchers should be aware of?

Researchers working with PSPC1 should be familiar with its molecular profile to accurately interpret experimental results. PSPC1 has a calculated molecular weight of 59 kDa based on its 523 amino acid sequence, but it is typically observed at approximately 66 kDa in gel electrophoresis . This discrepancy between calculated and observed molecular weights is important to note when analyzing Western blot results.

The gene encoding PSPC1 has the NCBI Gene ID 55269, with GenBank accession number BC014184 . The UniProt ID for PSPC1 is Q8WXF1 . This protein is expressed in various human, mouse, and rat tissues, making it accessible for study across different model systems . PSPC1 is characterized as a nuclear protein that participates in the formation of paraspeckles, which are subnuclear structures involved in the regulation of gene expression through the control of RNA processing .

Understanding these molecular characteristics is essential for experimental design, particularly for techniques like Western blotting where protein identification relies on molecular weight determination, and for genetic studies that might require sequence information for primer design or gene expression analysis.

How is PSPC1 involved in post-transcriptional gene regulation and RNA processing?

PSPC1 plays a sophisticated role in post-transcriptional gene regulation through multiple mechanisms. As an RNA-binding protein that interacts with poly(A), poly(G), and poly(U) RNA homopolymers, PSPC1 participates in various aspects of RNA metabolism . Research has shown that PSPC1 forms complexes with other DBHS family proteins including PSF (SFPQ) to regulate gene expression at the post-transcriptional level .

In ER-positive breast cancer, PSPC1 has been demonstrated to interact with PSF and participate in the post-transcriptional regulation of PSF target genes, including ESR1 and SCFD2 . This regulatory mechanism was elucidated through a series of immunoprecipitation, RNA-immunoprecipitation (RIP), and quantitative PCR (qPCR) experiments . The interaction between PSPC1 and its RNA targets is critical for understanding its function in different cellular contexts.

Additionally, PSPC1's role in paraspeckle formation is intrinsically linked to RNA processing. Paraspeckles are nuclear bodies formed around the long non-coding RNA Neat1, and PSPC1 is one of the core protein components of these structures . Research using knock-down approaches has shown that depletion of p54nrb, PSPC1, or Neat1 results in decreased FGF1 IRES activity and reduced endogenous FGF1 expression . This suggests that PSPC1 and other paraspeckle components may regulate internal ribosome entry site (IRES)-dependent translation, adding another layer to its role in post-transcriptional gene regulation.

What is the significance of PSPC1 in cancer research, particularly breast cancer?

PSPC1 has emerged as a significant factor in cancer research, particularly in hormone-dependent breast cancer. Studies have demonstrated that PSPC1 is associated with poor prognosis in ER-positive breast cancer patients . This clinical relevance is supported by mechanistic studies showing that siRNA-mediated PSPC1 silencing suppresses the proliferation of ER-positive breast cancer cells .

At the molecular level, PSPC1 exerts its effects through post-transcriptional regulation of key genes. Immunohistochemical analysis has revealed that strong immunoreactivity (IR) of PSPC1 correlates with poor prognosis for ER-positive breast cancer patients . Furthermore, PSPC1 interacts with PSF to regulate PSF target genes, including ESR1 (which encodes estrogen receptor alpha) and SCFD2 . The downstream effects of this regulation are significant, as strong SCFD2 immunoreactivity also correlates with poor prognosis, and combinations of PSPC1, PSF, and SCFD2 immunoreactivity serve as potent prognostic factors .

The PSPC1-SCFD2 axis has been identified as a potential therapeutic target in breast cancer management. Microarray analysis has identified DDIAS and MYBL1 as SCFD2 downstream target genes, and SCFD2 silencing has been shown to suppress tamoxifen-resistant breast tumor growth in vivo . These findings highlight the potential of targeting PSPC1 and its associated pathways in developing novel therapeutic approaches for endocrine therapy-resistant breast cancer.

How does PSPC1 interact with long non-coding RNA Neat1 in paraspeckle formation?

The interaction between PSPC1 and the long non-coding RNA Neat1 is fundamental to paraspeckle formation and function. Neat1 serves as an architectural RNA that provides the scaffold for paraspeckle assembly, while PSPC1 is one of the core protein components of these nuclear structures . Research has shown that the knock-down of paraspeckle components, including p54nrb, PSPC1, or Neat1 itself, leads to a decrease in FGF1 IRES activity and endogenous FGF1 expression .

Several factors may explain these context-dependent effects, including differences between human and mouse systems, cell type-specific regulation of IRES-dependent translation, and variations in gene structure (such as the additional upstream promoter and second IRES in human MYC) . Understanding these complex interactions requires careful experimental design that takes into account the specific cellular context and the precise mechanisms being investigated.

What methodological approaches are recommended for studying PSPC1 in the context of innate immune responses?

To investigate PSPC1's role in innate immune responses, researchers should employ a multi-faceted approach that addresses its participation in the HDP-RNP complex and the cGAS-STING pathway. PSPC1 has been shown to play a role in the regulation of DNA virus-mediated innate immune response by assembling into the HDP-RNP complex, which serves as a platform for IRF3 phosphorylation and subsequent immune activation .

For protein-protein interaction studies, immunoprecipitation techniques have been successfully employed to study PSPC1 interactions. The Proteintech antibody has been validated for immunoprecipitation using 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate , while the Abcam antibody has been used with 0.5mg HEK293 whole cell extract, 5μg of antibody, and 50μl of protein G magnetic beads .

To dissect the role of PSPC1 in signaling pathways, RNA interference approaches have proven effective. siRNA-mediated knockdown of PSPC1 has been used to demonstrate its functional importance in cell proliferation . For more comprehensive studies, researchers might consider combining knockdown approaches with phosphoproteomic analysis to track changes in signaling cascade activation.

Immunofluorescence techniques are valuable for studying the subcellular localization of PSPC1 during immune responses. The recommended dilution for immunofluorescence is 1:50-1:500 , with validation in cell lines such as PC-3 . When studying PSPC1 in the context of viral infection, researchers should consider time-course experiments to capture dynamic changes in PSPC1 localization and complex formation during different phases of the immune response.

What experimental controls and validation steps are essential when working with PSPC1 antibodies?

When working with PSPC1 antibodies, implementing rigorous controls and validation steps is crucial for ensuring reliable and reproducible results. First, researchers should verify antibody specificity using positive and negative controls. The literature indicates that HEK-293 cells, HepG2 cells, and PC-3 cells have been validated as positive controls for Western blot detection of PSPC1 . For immunoprecipitation, HEK-293 cells serve as appropriate positive controls .

Knockout or knockdown validation is a gold standard approach for antibody validation. Published applications have reported PSPC1 knockdown/knockout validation, with at least one publication specifically focusing on this approach . When possible, researchers should include PSPC1-depleted samples as negative controls to confirm antibody specificity.

For Western blot applications, it's important to note that PSPC1 has a calculated molecular weight of 59 kDa (523 amino acids) but is typically observed at approximately 66 kDa . This discrepancy should be considered when interpreting bands, and molecular weight markers should always be included.

When performing immunofluorescence, appropriate controls include secondary antibody-only samples to assess background staining, and competitive blocking with the immunizing peptide when available. Co-localization studies with other known paraspeckle markers such as p54nrb can provide additional validation of specific staining patterns.

How can researchers optimize PSPC1 detection in challenging samples or tissues?

Optimizing PSPC1 detection in challenging samples requires a systematic approach to sample preparation and antibody application. For protein extraction, researchers should consider that PSPC1 is predominantly nuclear and associated with paraspeckles . Therefore, nuclear extraction protocols that effectively solubilize nuclear proteins while preserving their native state are recommended. The addition of RNase inhibitors may be beneficial since PSPC1 is an RNA-binding protein and its interactions with RNA could affect epitope accessibility .

For Western blot applications in difficult tissues, transfer conditions may need optimization. Given PSPC1's observed molecular weight of 66 kDa , semi-dry transfer systems with methanol-containing buffers often provide good results. If background is problematic, increasing blocking time (overnight at 4°C) and using 5% BSA instead of milk for blocking and antibody dilution can improve signal-to-noise ratios.

For immunohistochemistry and immunofluorescence, antigen retrieval methods significantly impact PSPC1 detection. Heat-induced epitope retrieval using citrate buffer (pH 6.0) is often effective for formalin-fixed tissues . For particularly challenging samples, a step-wise dilution series (e.g., 1:50, 1:100, 1:200, 1:500) should be tested to determine the optimal antibody concentration .

Signal amplification systems such as tyramide signal amplification (TSA) or polymer-based detection systems can enhance sensitivity in tissues with low PSPC1 expression. Additionally, co-staining with markers of nuclear paraspeckles can help confirm specific PSPC1 detection and localization in complex tissue samples.

What are the potential pitfalls in interpreting PSPC1 experimental data and how can they be avoided?

Interpreting PSPC1 experimental data requires awareness of several potential pitfalls. First, the discrepancy between PSPC1's calculated molecular weight (59 kDa) and its observed migration on SDS-PAGE (66 kDa) might lead to misidentification . Researchers should always include molecular weight markers and consider post-translational modifications that might affect migration patterns.

Subcellular localization interpretation can be complicated by PSPC1's dynamic distribution. While primarily nuclear and concentrated in paraspeckles, PSPC1 distribution may change under different cellular conditions . Using proper nuclear and cytoplasmic markers in co-staining experiments helps avoid misinterpretation of localization patterns.

When studying PSPC1's role in cancer, particularly breast cancer, sample heterogeneity can confound results. Patient-derived samples may exhibit variable PSPC1 expression depending on tumor subtype, stage, and previous treatments . Stratifying samples based on clinicopathological parameters (such as ER status) and using sufficiently large cohorts are essential for meaningful correlations .

Finally, context-dependency of PSPC1 function across different cell types can lead to apparently contradictory results . Researchers should be cautious when extrapolating findings across different cellular systems and should explicitly state the specific cell types and conditions used in their experiments.

What are the emerging research directions for PSPC1 antibody applications?

PSPC1 research is expanding beyond its traditional role in paraspeckle biology to encompass broader implications in disease mechanisms and potential therapeutic applications. The association of PSPC1 with poor prognosis in ER-positive breast cancer has opened new avenues for cancer biomarker development and therapeutic targeting . Future research will likely focus on developing more specific tools to manipulate PSPC1 function in cancer cells, potentially including domain-specific antibodies that can distinguish between different functional states of PSPC1.

The role of PSPC1 in innate immune responses through the HDP-RNP complex suggests potential applications in immunology research . Developing antibodies that specifically recognize PSPC1 within different protein complexes could help delineate its context-specific functions in immune signaling pathways. Additionally, exploring PSPC1's involvement in the circadian clock regulation may lead to novel applications in chronobiology research .

Technical advances are likely to include the development of phospho-specific PSPC1 antibodies to monitor its activation state, and conformational antibodies that distinguish between RNA-bound and unbound forms. The integration of PSPC1 antibodies into multiplexed detection systems, including mass cytometry and multiplexed immunofluorescence, will enable more comprehensive analysis of PSPC1 in the context of complex cellular networks.

Finally, as therapeutic applications targeting RNA-binding proteins continue to develop, PSPC1 antibodies may find utility in validating drug mechanisms and monitoring treatment responses in preclinical models, particularly for hormone-dependent cancers where PSPC1 has demonstrated prognostic significance .

How can researchers integrate PSPC1 studies into broader investigations of nuclear organization and gene regulation?

Integrating PSPC1 studies into broader investigations of nuclear organization and gene regulation requires a multidisciplinary approach that places PSPC1 within the context of dynamic nuclear architecture. As a paraspeckle component, PSPC1 offers a window into the organization and function of these nuclear bodies . Combining PSPC1 antibody-based approaches with advanced imaging techniques such as super-resolution microscopy and live-cell imaging can provide insights into the dynamic assembly and disassembly of paraspeckles in response to cellular stresses and developmental cues.

The interaction between PSPC1 and long non-coding RNA Neat1 highlights the importance of RNA-protein interactions in nuclear organization . Researchers can leverage this by combining PSPC1 immunoprecipitation with RNA-sequencing approaches to identify the complete repertoire of RNAs associated with PSPC1 in different cellular contexts. Similarly, chromatin immunoprecipitation followed by sequencing (ChIP-seq) can map PSPC1's genomic binding sites, illuminating its direct role in transcriptional regulation.

The identification of PSPC1 as a regulator of post-transcriptional processes, including IRES-dependent translation , suggests that PSPC1 participates in coordinating nuclear events with cytoplasmic gene expression. Researchers can investigate this coordination using subcellular fractionation combined with proteomic and transcriptomic analyses to track PSPC1-associated complexes across cellular compartments.