PRKCSH Antibody

What is PRKCSH and why is it important in biological research?

PRKCSH (Protein Kinase C Substrate 80K-H) encodes the beta-subunit of glucosidase II, a critical N-linked glycan-processing enzyme in the endoplasmic reticulum (ER) quality control system. This protein is essential for identifying and eliminating misfolded proteins, ensuring proper protein folding and release from the ER. PRKCSH has gained significant research interest due to its involvement in various cellular processes including cancer development, glycosylation events, and its role in the unfolded protein response (UPR) . The gene is located on chromosome 19p13.2, and mutations in PRKCSH are associated with autosomal dominant polycystic liver disease (ADPLD) .

What are the known alternative names and isoforms of PRKCSH?

PRKCSH is known by several aliases including:

• Glucosidase II beta subunit (GluIIβ)

• 80K-H

• Hepatocystin

• PCLD/PCLD1 (polycystic liver disease)

• PLD1 (phospholipase D1, phosphatidylcholine-specific)

• G19P1

• AGE-R2 (advanced glycation end product receptor-2)

• PKCSH

• VASAP-60

Multiple alternatively spliced transcript variants have been documented, resulting in different isoforms of PRKCSH with distinct functions, particularly in processes like epithelial-mesenchymal transition (EMT) .

What applications are PRKCSH antibodies typically used for?

Note: It is recommended that antibodies be titrated in each testing system to obtain optimal results, as performance can be sample-dependent .

How does PRKCSH contribute to cancer development and progression?

PRKCSH's role in cancer is multifaceted and involves several mechanisms:

• TNFSF Resistance: PRKCSH abundance in lung cancers enhances oncogenic IGF1R activation by extending its half-life, leading to resistance to tumor necrosis factor superfamily (TNFSF) signaling. This impairs caspase-8 activation, increases Mcl-1 expression, and inhibits caspase-9, creating an imbalance between cell death and survival pathways .

• IRE1α Signaling: PRKCSH functions as a selective activator of the IRE1α branch of the unfolded protein response (UPR). It boosts ER stress-mediated autophosphorylation and oligomerization of IRE1α through direct interaction, contributing to tumor cell adaptation to stress and tumorigenesis .

• N-linked Glycosylation: PRKCSH assists cancer cells in managing increased demand for N-linked glycoproteins and ER stress. This supports cancer cell growth by promoting proper folding of growth factor receptors like EGFR and TGF-β receptors, which play critical roles in regulating cell proliferation .

• Expression Correlation: Elevated PRKCSH expression has been documented across various cancer tissues including esophageal carcinoma, glioblastoma, liver hepatocellular carcinoma, lymphoid neoplasm, thymoma, pancreatic adenocarcinoma, skin cutaneous melanoma, and stomach adenocarcinoma. High expression correlates with extrahepatic metastasis, advanced TNM stage, and poor survival rates .

What is the relationship between PRKCSH and the unfolded protein response in research models?

PRKCSH has been identified as a selective activator of the IRE1α branch of the unfolded protein response. Research has demonstrated that:

• PRKCSH enhances IRE1α signaling pathway by promoting autophosphorylation and oligomerization of IRE1α through direct interaction specifically under ER stress conditions .

• It exhibits a dual function: modulating glycoprotein quality control under normal conditions and activating the IRE1α-mediated stress response pathway upon ER stress through domain-specific interactions .

• The IRE1α pathway activated by PRKCSH sustains microsomal prostaglandin E synthase-1 (mPGES-1) expression, leading to the production of immunosuppressive prostaglandin E2. This mechanism facilitates the advancement of non-small cell lung cancer (NSCLC) by compromising the ability of adaptive immune cells in the tumor microenvironment to effectively destroy tumor cells .

• In experimental models, suppressing PRKCSH led to a dose-dependent down-regulation of EGFR/RTK and PI3K/AKT signaling activities, resulting in significantly reduced growth rates, particularly in conditions of low nutrient availability .

How can circular RNA PRKCSH be detected and what is its significance?

Circular RNA PRKCSH (circPrkcsh) represents an emerging area of research with specific detection methods:

• Detection Method: Fluorescence in situ hybridization (FISH) assays using circPrkcsh-specific probes have been employed to detect circPrkcsh in primary mouse astrocytes. In the protocol, 4′,6-Diamidino-2-phenylindole (blue) is used to label nuclei, while Cy-3 (red) labels circPrkcsh probes .

• Significance in Inflammatory Response: Research has revealed elevated expression of circPrkcsh with a concurrent decrease in miR-488 expression in injured cells. CircPrkcsh regulates the expression of the inflammation-related gene Ccl2 and may function as an miR-488 sponge .

• Therapeutic Potential: In tumor necrosis factor-α-treated astrocytes, circPrkcsh knockdown decreased the expression of Ccl2 by upregulating miR-488 expression and reduced the secretion of inflammatory cytokines, suggesting potential therapeutic applications in spinal cord injury .

What are the optimal sample preparation techniques for PRKCSH antibody experiments?

For optimal results with PRKCSH antibody experiments, consider the following sample preparation techniques:

• Western Blot Sample Preparation:

  • Use 25μg protein per lane for cell/tissue lysates

  • For blocking, 3-5% nonfat dry milk in TBST is effective

  • When detecting PRKCSH, brief exposure times (5-10 seconds) with ECL detection systems are often sufficient

• Immunoprecipitation:

  • Use 0.5-4.0 μg antibody for every 1.0-3.0 mg of total protein lysate

  • HeLa cells have been validated as positive controls for IP experiments

• Immunohistochemistry:

  • For antigen retrieval, TE buffer pH 9.0 is recommended

  • Alternatively, citrate buffer pH 6.0 can be used

  • Human kidney tissue and normal colon have been validated as positive controls

• Cell Culture Models:

  • A431, HeLa, and Jurkat cells have been validated for PRKCSH expression

  • For tissue models, mouse and rat liver tissues show consistent PRKCSH expression

How can PRKCSH knockdown experiments be designed and validated?

For effective PRKCSH knockdown experiments:

• siRNA Design: Small interfering RNA (siRNA) duplexes targeting different PRKCSH splice variants have been successfully used. For the two major alternative splicing transcripts, the following target sequences have been validated:

  • PRKCSH-1: 5'...ATGGAGAACCAAGGGACACG...3' and 5'...CTAAAAATTAAATCCAAAGC...3'

  • PRKCSH-2: 5'...ATGGTTTTTAGTATCCAAGA...3' and 5'...CTAAAAATTAAATCCAAAGC...3'

• Transfection Protocol: Transfect 4nM siRNA duplexes using Lipofectamine RNAiMAX (or equivalent transfection reagent) following manufacturer's instructions .

• Validation Methods:

  • Western blot analysis using PRKCSH-specific antibodies (dilution 1:500-1:1000)

  • Quantitative RT-PCR to confirm reduction in PRKCSH mRNA levels

  • Functional validation through monitoring downstream effects such as changes in EMT markers (E-cadherin, N-cadherin, vimentin, and α-SMA)

• Knockdown Effect Assessment:

  • Cell proliferation assays using Cell Counting Kit-8 (CCK-8)

  • Wound healing experiments to assess migration capacity

  • Analysis of EMT-related biomarkers by western blot

What control samples should be included when using PRKCSH antibodies?

To ensure experimental validity with PRKCSH antibodies, include these controls:

• Positive Controls for Western Blot:

  • A431 cells, HeLa cells, Jurkat cells (human)

  • Mouse liver tissue, rat liver tissue (rodent)

• Positive Controls for Immunoprecipitation:

  • HeLa cell lysates

• Positive Controls for Immunohistochemistry:

  • Human kidney tissue

  • Human normal colon tissue

• Positive Controls for Immunofluorescence:

  • HeLa cells

• Negative Controls:

  • Secondary antibody-only controls to assess non-specific binding

  • PRKCSH-knockout or knockdown samples (if available)

  • Isotype controls using non-specific IgG of the same species and isotype

• Experimental Validation:

  • When studying PRKCSH in relation to IRE1α signaling, include samples treated with and without ER stress inducers (like tunicamycin or thapsigargin)

  • For cancer-related studies, pair normal and tumor tissue samples from the same origin

Why is there a discrepancy between calculated and observed molecular weights of PRKCSH?

The discrepancy between the calculated molecular weight (59 kDa) and the observed molecular weight (80 kDa) of PRKCSH in experimental settings can be attributed to several factors:

• Post-translational Modifications: PRKCSH undergoes extensive post-translational modifications, particularly phosphorylation. It contains four potential protein kinase C phosphorylation sites, which can significantly alter its migration pattern in SDS-PAGE .

• Glycosylation: As a protein involved in N-linked glycosylation processes, PRKCSH itself may be glycosylated, contributing to its increased apparent molecular weight .

• Structural Features: The protein exhibits a complex structure with several distinctive features, including an EF-hand domain for calcium binding and a significant glutamic acid repeat. Additionally, it has a polar distribution of cysteine residues suggesting the presence of multiple intra- and/or intermolecular disulfide bridges, which can affect its electrophoretic mobility .

• Isoform Variation: Multiple alternatively spliced transcript variants exist, potentially resulting in different isoforms with varying molecular weights .

When validating PRKCSH antibodies, it is important to account for this discrepancy and expect the protein to appear at approximately 80 kDa on western blots despite its calculated weight of 59 kDa .

How can researchers address cross-reactivity concerns with PRKCSH antibodies?

To address potential cross-reactivity issues with PRKCSH antibodies:

• Antibody Validation:

  • Verify antibody specificity using knockout or knockdown models

  • Test the antibody against multiple cell lines and tissue samples to confirm consistent detection patterns

  • Compare results from antibodies targeting different epitopes of PRKCSH

• Species Cross-Reactivity:

  • PRKCSH antibodies like 12148-1-AP have demonstrated reactivity with human, mouse, and rat samples

  • When using antibodies across species, validate by including appropriate positive controls from each species

  • Be aware that sequence homology may differ at specific epitopes across species

• Technical Approaches:

  • Use appropriate blocking buffers (3-5% nonfat dry milk or BSA in TBST)

  • Optimize antibody concentration through titration experiments (e.g., 1:2000-1:8000 for WB)

  • Include additional washing steps to reduce background

  • Consider pre-absorption with potential cross-reactive proteins

• Epitope Considerations:

  • Select antibodies that target unique regions of PRKCSH to minimize cross-reactivity

  • For example, the A0894 antibody uses a synthetic peptide corresponding to a sequence within amino acids 429-528 of human PRKCSH, which may offer specificity advantages

How should researchers interpret PRKCSH expression data in relation to cancer progression?

When interpreting PRKCSH expression data in cancer research:

• Expression Level Correlation: Multiple studies have shown that elevated PRKCSH expression correlates with poor prognosis across various cancer types. Specifically, high PRKCSH expression has been associated with extrahepatic metastasis, advanced TNM stage, and poor survival rates .

• Mechanistic Interpretation: Consider that PRKCSH may contribute to cancer progression through multiple mechanisms:

  • Enhanced IRE1α signaling, promoting tumor cell adaptation to stress

  • Extended IGF1R half-life, resulting in TNFSF resistance

  • Improved N-linked glycoprotein processing, supporting growth factor receptor function

  • Alternative splicing variants affecting EMT processes

• Context-Dependent Effects: PRKCSH expression should be interpreted in the context of:

  • The specific cancer type being studied

  • Expression of related proteins (e.g., IGF1R, IRE1α)

  • Tumor microenvironment factors

  • Patient clinical data

• Contradictory Data Resolution: When facing seemingly contradictory data regarding PRKCSH expression:

  • Examine which specific isoform of PRKCSH is being detected (different isoforms may have distinct functions)

  • Consider cell-type specificity (effects may differ between cell types)

  • Evaluate the experimental conditions (stress conditions may alter PRKCSH function)

  • Compare methods of detection (antibody-based versus RNA-based measurements)

• Bioinformatic Analysis: Studies utilizing TCGA data revealed that PRKCSH expression is elevated across multiple cancer types. Researchers should consider using similar bioinformatic approaches when analyzing their own PRKCSH expression data to place it in broader context .

How is PRKCSH being investigated in relation to anti-tumor immunity?

Recent research has revealed important connections between PRKCSH and anti-tumor immunity:

• IRE1α Pathway Modulation: PRKCSH directly activates the IRE1α signaling pathway, which has been shown to influence anti-tumor immunity. In non-small cell lung cancer (NSCLC), activation of this pathway sustains microsomal prostaglandin E synthase-1 (mPGES-1) expression, leading to the production of immunosuppressive prostaglandin E2. This mechanism compromises the ability of adaptive immune cells in the tumor microenvironment to effectively destroy tumor cells .

• NK Cell Activity Enhancement: PRKCSH deficiency has been shown to augment the antitumor effects of natural killer (NK) cells in a tumor xenograft IL-2Rg-deficient NOD/SCID (NIG) mouse model. This suggests that PRKCSH suppression could potentially enhance NK cell-based cancer therapies .

• T Cell Immunity: PRKCSH modulation affects T cell activity in the tumor microenvironment. Studies suggest that its suppression may enhance T cell activity, offering promising approaches for enhancing cancer immunotherapy .

• Experimental Approaches: Researchers investigating PRKCSH in relation to anti-tumor immunity typically employ:

  • Knockout or knockdown models of PRKCSH

  • Co-culture systems with immune effector cells

  • In vivo tumor models with intact or compromised immune systems

  • Analysis of immune cell infiltration and activity within the tumor microenvironment

What is the current understanding of PRKCSH's role in alternative splicing and EMT?

The relationship between PRKCSH alternative splicing and epithelial-mesenchymal transition (EMT) represents an emerging area of research:

• Alternative Splice Variants: Two major PRKCSH alternative splicing transcripts (PRKCSH-1 and PRKCSH-2) have been identified with different functions, particularly in regulating EMT progression. These distinct isoforms appear to have differential effects on cellular processes .

• Impact on Cell Proliferation: Research has demonstrated that PRKCSH-2 knockdown promotes A549 cell proliferation potential, partially by promoting EMT signals. This suggests isoform-specific functions in regulating cell growth and metastatic potential .

• Environmental Triggers: Silica exposure has been shown to affect alternative splicing of PRKCSH in lung cells. Bioinformatic analysis of silica-exposed cells revealed that genes with alternative splicing were mainly associated with EMT pathway, N-Glycan biosynthesis, and leukocyte transendothelial migration .

• Experimental Approaches: To study PRKCSH alternative splicing:

  • Use isoform-specific siRNAs targeting different splice variants

  • Monitor EMT markers (E-cadherin, N-cadherin, vimentin, α-SMA) following isoform-specific knockdown

  • Analyze cell proliferation and migration using wound healing assays

  • Employ TGF-β1 treatment (10 ng/mL) to induce EMT in experimental models

What research tools are being developed to better understand PRKCSH function?

Advanced research tools for studying PRKCSH include:

• CRISPR/Cas9 Knockout Models: Generation of PRKCSH-knockout cell lines and animal models to study functional consequences of complete PRKCSH loss .

• Isoform-Specific Knockdown: Development of siRNAs targeting specific PRKCSH splice variants allows for the study of isoform-specific functions:

  • PRKCSH-1: 5'...ATGGAGAACCAAGGGACACG...3' and 5'...CTAAAAATTAAATCCAAAGC...3'

  • PRKCSH-2: 5'...ATGGTTTTTAGTATCCAAGA...3' and 5'...CTAAAAATTAAATCCAAAGC...3'

• Domain-Specific Antibodies: Development of antibodies targeting specific domains of PRKCSH to study domain-specific interactions and functions.

• Fluorescence In Situ Hybridization (FISH): For detection of circular RNA PRKCSH (circPrkcsh), FISH assays with specific probes have been developed to study its subcellular localization and expression patterns .

• Live-Cell Imaging Approaches: To study PRKCSH translocation in response to stimuli such as acidic fibroblast growth factor (aFGF/FGF-1) or basic fibroblast growth factor (bFGF/FGF-2), which have been shown to induce phosphorylation and subcellular redistribution of PRKCSH .

• Interaction Studies: Methods to investigate PRKCSH interactions with partners like IGF1R and IRE1α include:

  • Co-immunoprecipitation

  • Proximity ligation assays

  • FRET/BRET systems for detecting protein-protein interactions in living cells

What are the emerging therapeutic implications of PRKCSH research?

PRKCSH research suggests several promising therapeutic directions:

• Cancer Therapy Enhancement: PRKCSH suppression may enhance the efficacy of natural killer (NK) cell-based cancer therapies and potentially improve responses to other immunotherapeutic approaches .

• ER Stress Modulation: As a regulator of the IRE1α branch of the unfolded protein response, PRKCSH presents a potential target for modulating ER stress responses in cancer cells, potentially sensitizing them to therapeutic interventions .

• Anti-inflammatory Applications: In neurological contexts, targeting circPrkcsh has shown promise in reducing inflammatory responses after spinal cord injury, suggesting potential therapeutic applications in neuroinflammatory conditions .

• Glycosylation Pathway Interventions: PRKCSH's role in N-linked glycan processing suggests that modulating its activity could affect glycosylation patterns of cancer-associated proteins, potentially altering their function and stability .

• Isoform-Specific Approaches: The discovery of functionally distinct PRKCSH isoforms opens possibilities for targeting specific variants that contribute to pathological processes while sparing those with normal physiological functions .