REG4 Human, Sf9

What is REG4 and what are its key structural characteristics?

REG4 is a member of the regenerating gene family first identified in ulcerative colitis cDNA libraries. The human REG4 produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain spanning amino acids 23-158 and is typically fused to a 9 amino acid His-Tag at the C-terminus. The complete construct contains 145 amino acids with a molecular mass of approximately 17kDa . On SDS-PAGE under reducing conditions, REG4 typically displays multiple bands between 13.5-18kDa, reflecting its glycosylation status. The protein belongs to the REG family that includes REG 1 alpha, REG 1 beta, REG-related sequence (RS), and HIP/PAP, all sharing similar structural features despite being classified into different subfamilies based on amino acid sequence variations .

How does REG4 Human, Sf9 recombinant protein differ from native human REG4?

The recombinant REG4 Human produced in Sf9 cells contains several modifications that distinguish it from native human REG4. The recombinant version is expressed without the 22-amino acid signal peptide (starting at position 23) and includes a C-terminal His-tag fusion for purification purposes . While the core protein structure maintains the critical functional domains of native REG4, these modifications optimize the protein for laboratory research applications. Additionally, the glycosylation pattern in insect Sf9 cells differs from mammalian cells, potentially affecting certain protein properties while maintaining core biological activity for research purposes .

What are the optimal storage conditions for REG4 Human, Sf9 to maintain stability?

For optimal stability of REG4 Human, Sf9 recombinant protein, a tiered storage approach is recommended depending on usage timeframe. For short-term use (within 2-4 weeks), the protein can be stored at 4°C in its original formulation. For medium to long-term storage, maintain the protein at -20°C . Research shows that adding a carrier protein (0.1% HSA or BSA) significantly enhances long-term stability by preventing protein adsorption to container surfaces and providing cryoprotection . Multiple freeze-thaw cycles must be strictly avoided as they promote protein denaturation and aggregation, potentially compromising both structural integrity and biological activity. When working with the protein, always maintain sterile conditions to prevent microbial contamination .

How should REG4 Human, Sf9 be incorporated into cell culture experiments?

When designing cell culture experiments with REG4 Human, Sf9, researchers should consider several methodological factors. First, determine the appropriate concentration range based on experimental objectives—typically starting with 10-100 ng/mL for signaling studies and 100-500 ng/mL for functional assays. Due to REG4's role in activating EGFR/Akt pathways, use serum-free or low-serum conditions (0.5-1% FBS) during treatment periods to minimize interference from serum growth factors .

For treatment protocols, pre-incubation of cells in reduced serum for 6-12 hours before adding REG4 improves signal-to-noise ratio. When studying REG4's effects on cancer cell lines, consider the baseline expression levels of potential REG4 receptors (CD44, GPR37) and pathway components as these significantly impact cellular response magnitudes. Include appropriate controls, including untreated cells and cells treated with a non-functional protein at equivalent concentrations. Time-course experiments (30 minutes to 72 hours) are essential to distinguish between immediate signaling events and downstream transcriptional responses .

What analytical techniques are most effective for studying REG4-induced signaling pathways?

For comprehensive analysis of REG4-induced signaling, a multi-platform approach is recommended. Western blotting remains the primary method for detecting phosphorylation events in the EGFR/Akt/AP-1 and Akt/GSK3β/β-catenin/TCF-4 pathways, with particular attention to phosphorylation sites Tyr992 and Tyr1068 on EGFR, and Thr308 and Ser473 on Akt . For temporal dynamics of pathway activation, phospho-specific flow cytometry offers single-cell resolution with higher throughput.

To assess transcriptional outcomes, quantitative RT-PCR targeting downstream genes like Bcl-xL, Bcl-2, survivin, and MMP-7 provides reliable quantitation. For unbiased discovery of REG4-regulated genes, RNA-seq with differential expression analysis at multiple time points (6, 12, 24 hours) captures both early and late transcriptional responses. Transcription factor activity assays specific for AP-1 (particularly JunB, JunD, and FosB) and TCF-4 directly measure REG4's effect on transcriptional activation . For protein-protein interactions, co-immunoprecipitation assays followed by mass spectrometry can identify novel REG4 binding partners beyond known interactions with CD44, GPR37, mannan, and heparin .

How does REG4 contribute to cancer progression at the molecular level?

REG4 promotes cancer progression through multiple coordinated molecular mechanisms. At the cellular signaling level, REG4 activates EGFR by inducing phosphorylation at specific tyrosine residues (Tyr992 and Tyr1068), which triggers downstream Akt phosphorylation (at Thr308 and Ser473) . This activation initiates two critical pathways: the EGFR/Akt/AP-1 axis and the Akt/GSK3β/β-catenin/TCF-4 cascade .

In the first pathway, activated AP-1 transcription factors (specifically JunB, JunD, and FosB) upregulate anti-apoptotic proteins including Bcl-xL, Bcl-2, and survivin, conferring resistance to programmed cell death . Simultaneously, matrix metalloproteinase-7 (MMP-7) expression increases, enhancing invasive capacity . The second pathway involves inhibitory phosphorylation of GSK3β, stabilizing β-catenin, which translocates to the nucleus and partners with TCF-4 to promote cell cycle progression, particularly at the G2 phase .

Additionally, REG4 interacts with CD44, activating its regulated intramembrane proteolysis. This process releases the CD44 intracytoplasmic domain (CD44ICD), which functions as a transcriptional activator for D-type cyclins (promoting proliferation) and stem cell factors including Kruppel-like factor 4 and SOX2 (enhancing cancer stemness) .

What is the relationship between REG4 expression and clinical outcomes in cancer patients?

Clinical research demonstrates significant correlations between REG4 expression and patient outcomes across multiple cancer types. Overexpression of REG4 has been consistently documented in gastric, colorectal, pancreatic, gallbladder, ovarian, and urothelial cancers, with elevated expression strongly associated with aggressive disease phenotypes and poor prognosis .

In gastric cancer, REG4 expression correlates significantly with both intestinal mucin phenotype (marked by MUC2 and CDX2 expression) and neuroendocrine differentiation . Histopathologically, REG4 is specifically expressed in neuroendocrine tumors and signet ring cell carcinomas of the gastrointestinal tract, pancreas, ovary, and lung, making it a potential diagnostic marker for these subtypes .

Notably, REG4 expression appears to support a proposed histogenetic sequence in gastric cancer: gastric intestinal metaplasia → globoid dysplasia → signet ring cell carcinoma . The mechanistic link between REG4 expression and poor clinical outcomes stems from its promotion of multiple cancer hallmarks: enhanced proliferation, apoptosis resistance, chemoradiotherapy resistance, increased migration and invasion capacity, peritoneal dissemination capability, accelerated tumor growth, and maintenance of cancer stem cell properties .

How do transcriptional and translational regulators control REG4 expression?

REG4 expression is regulated through multiple sophisticated transcriptional and translational control mechanisms. At the transcriptional level, several transcription factors directly activate REG4 expression by binding to specific promoter elements. Caudal type homeobox 2 (CDX2) induces REG4 expression by binding to consensus CDX2-binding elements upstream of the REG4 gene, supported by the positive correlation between CDX2 and REG4 expression in gastric cancer tissues .

GATA binding protein 6 (GATA6) functions as another critical transcriptional activator of REG4, particularly in colon cancer cells where it also induces Lgr5 expression essential for growth under adherent conditions . The Hedgehog signaling pathway contributes to REG4 regulation through GLI family zinc finger 1 (GLI1), which binds directly to REG4 promoter regions (specifically the GATCATCCA sequence) to enhance transcription .

Additional transcriptional regulators include activating transcription factor 2 (ATF2), which targets the REG4 promoter during enteritis, and specificity protein 1 (SP1), which can be stimulated by TGF-α to transcriptionally promote REG4 expression . Interestingly, SRY-box transcription factor 9 (SOX9) appears to have a complex regulatory relationship with REG4, as SOX9 knockdown upregulates REG4 protein expression in gastric cancer cells .

At the translational level, microRNA-24 (miR-24) has been identified as a tumor suppressor that translationally restrains gastric cancer progression by down-regulating REG4 . This multi-layered regulatory network allows for context-specific expression of REG4 in different tissues and disease states.

What are the methodological approaches for investigating REG4 protein-protein interactions?

Investigating REG4 protein-protein interactions requires sophisticated methodological approaches tailored to this secreted glycoprotein. For in vitro analysis, surface plasmon resonance (SPR) with immobilized REG4 or potential binding partners can quantitatively measure binding kinetics and affinities with purified proteins. When studying cell surface interactions, crosslinking studies using membrane-impermeable crosslinkers followed by immunoprecipitation and mass spectrometry can identify physiologically relevant membrane protein interactions.

For comprehensive interactome analysis, BioID or APEX2 proximity labeling systems can be particularly valuable—REG4 fusion constructs with these biotin ligases identify proteins in close proximity under physiological conditions. To validate specific interactions with known partners like CD44, GPR37, or glycan structures (mannan and heparin), mutagenesis of predicted interaction domains followed by co-immunoprecipitation or binding assays can determine essential residues for interaction .

For visualizing interactions in cellular contexts, proximity ligation assays (PLA) or fluorescence resonance energy transfer (FRET) provide spatial information about REG4 interactions. To address the glycosylation-dependent interactions of REG4, comparing binding profiles of glycosylated Sf9-produced REG4 with enzymatically deglycosylated protein or bacterially expressed non-glycosylated REG4 can reveal glycan-dependent interactions. These methodological approaches, used in combination, can provide comprehensive insights into the REG4 interactome and its functional significance .

How can contradictory findings about REG4 functions be reconciled in experimental design?

Reconciling contradictory findings regarding REG4 functions requires methodological sophistication in experimental design. One fundamental approach is conducting parallel experiments in multiple cellular contexts, as REG4's effects appear highly context-dependent. Researchers should systematically compare REG4 functions across different cell types (epithelial vs. mesenchymal, primary vs. immortalized, normal vs. cancer) to identify cell-type specific responses .

The expression levels of REG4 receptors (CD44, GPR37) and downstream signaling components should be precisely characterized in each experimental system, as receptor density dramatically influences response magnitude and pathway selection. Dose-response experiments across a wide concentration range (1-1000 ng/mL) can reveal biphasic effects that may explain apparently contradictory results obtained at single concentrations .

Temporal dynamics analysis is crucial, as REG4 may elicit different or even opposing effects at different time points post-treatment. Short-term (minutes to hours) versus long-term (days to weeks) experiments should be conducted in parallel. For in vivo studies, genetic background significantly impacts REG4 biology, necessitating the use of multiple mouse strains when evaluating REG4 functions in transgenic or knockout models .

What are the emerging therapeutic strategies targeting REG4 in cancer treatment?

Emerging therapeutic strategies targeting REG4 in cancer treatment are developing along several promising avenues. Direct REG4 neutralization approaches include humanized monoclonal antibodies that bind REG4 and prevent its interaction with receptors. These antibodies are being engineered with enhanced tumor penetration properties and reduced immunogenicity. Small molecule inhibitors that disrupt specific protein-protein interactions between REG4 and its binding partners (particularly CD44 and GPR37) are under development, with structure-based drug design guiding optimization efforts .

Receptor-targeted approaches focus on CD44 and GPR37, employing antibody-drug conjugates that specifically deliver cytotoxic payloads to REG4-responsive cancer cells. Dual-targeting bispecific antibodies that simultaneously engage REG4 and its receptors are being designed to enhance binding specificity and therapeutic efficacy .

Pathway-focused strategies target downstream signaling, with several Akt inhibitors in clinical trials showing enhanced efficacy in REG4-expressing tumors. Combination therapies pairing REG4 pathway inhibitors with conventional chemotherapeutics are being explored to overcome the chemoresistance conferred by REG4 expression .

RNA interference approaches using lipid nanoparticle-delivered siRNA targeting REG4 mRNA have shown promising results in preclinical models. For transcriptional regulation, small molecules targeting the transcription factors that drive REG4 expression (particularly CDX2 and GATA6) are in early development stages. These multi-faceted approaches reflect the growing recognition of REG4 as a significant therapeutic target in multiple cancer types .

What are the critical considerations when designing experiments to study REG4-induced cellular changes?

When designing experiments to study REG4-induced cellular changes, researchers must address several critical considerations to ensure reliable and reproducible results. Cell line selection requires careful attention to baseline expression levels of REG4, its receptors (CD44, GPR37), and components of downstream signaling pathways (EGFR, Akt). Ideally, experiments should include multiple cell lines representing different levels of endogenous REG4 expression to capture the spectrum of potential responses .

For treatment protocols, recombinant REG4 concentration is crucial—starting with 10-500 ng/mL while including dose-response curves to identify potential concentration-dependent effects. Time course design should encompass both rapid signaling events (minutes to hours) and longer-term transcriptional and phenotypic changes (24-72 hours) .

Control conditions must include vehicle controls matched precisely to the REG4 formulation (containing matching buffer components and carrier proteins) and irrelevant protein controls (ideally a structurally similar protein at equimolar concentration). For knockdown or knockout studies, multiple siRNA/shRNA sequences or different CRISPR guide RNAs should be employed to rule out off-target effects .

Pathway analysis requires selective inhibitors targeting key nodes (EGFR, Akt, GSK3β) to delineate the contribution of specific pathways to observed phenotypes. When measuring biological outcomes, complementary assays should be used for critical endpoints—for example, assessing proliferation by both metabolic activity (MTT/XTT) and direct cell counting or DNA synthesis (BrdU/EdU incorporation) .

What troubleshooting strategies can address common challenges when working with REG4 Human, Sf9?

When working with REG4 Human, Sf9, researchers frequently encounter several technical challenges that require systematic troubleshooting approaches. For protein stability issues, if activity decreases during storage, implement more stringent temperature control and consider aliquoting the protein in single-use volumes to avoid freeze-thaw cycles. Adding carrier proteins (0.1% HSA or BSA) can significantly enhance stability, particularly for dilute solutions .

For inconsistent cellular responses, first verify protein activity using a validated positive control assay, such as EGFR phosphorylation in a responsive cell line like colorectal cancer cells. Cell culture conditions significantly impact REG4 responsiveness—ensure serum starvation before treatment (typically 6-12 hours in 0.5-1% serum) to reduce background pathway activation .

When facing detection difficulties in binding or interaction studies, optimize primary antibody conditions and consider epitope accessibility issues that may arise from REG4's glycosylation pattern. For co-immunoprecipitation experiments, try different lysis buffers as REG4 interactions may be sensitive to detergent type and concentration .

For pathway analysis challenges, phospho-specific antibodies may require optimization of fixation and permeabilization protocols. If downstream targets show minimal regulation, verify the functional integrity of the pathway using positive control stimuli (EGF for EGFR/Akt pathway, Wnt ligands for β-catenin pathway) .

When glycosylation heterogeneity interferes with experiments, consider enzymatic deglycosylation under native conditions or compare results with non-glycosylated recombinant REG4 produced in bacterial systems. These systematic troubleshooting approaches can address most common challenges encountered when working with REG4 Human, Sf9 .

What statistical approaches are appropriate for analyzing REG4 experimental data?

Analyzing REG4 experimental data requires tailored statistical approaches depending on the experimental design and data characteristics. For comparative studies examining REG4 effects across different conditions or cell lines, analysis of variance (ANOVA) with appropriate post-hoc tests (Tukey's or Dunnett's) should be applied when comparing multiple groups. For dose-response relationships, nonlinear regression analysis using four-parameter logistic models can accurately determine EC50 values and maximum response magnitudes .

Time-course experiments benefit from repeated measures ANOVA or mixed-effects models that account for the correlated nature of measurements from the same experimental units over time. When analyzing REG4's effects on signaling pathways, integrated pathway analysis using principal component analysis or partial least squares discriminant analysis can identify coordinated changes across multiple pathway components .

For survival analysis in animal models or clinical datasets, Kaplan-Meier curves with log-rank tests stratified by REG4 expression levels provide insights into prognostic significance. Cox proportional hazards regression should be used to adjust for covariates and determine independent prognostic value. When analyzing gene expression correlations with REG4, Pearson or Spearman correlation coefficients are appropriate depending on data distribution characteristics .

Sample size calculations should account for the typically high biological variability in REG4 responses—power analyses with effect sizes estimated from preliminary data ensure adequate statistical power. For all analyses, researchers should report exact p-values, confidence intervals, and effect sizes rather than simply indicating statistical significance thresholds .

How can researchers differentiate between direct and indirect effects of REG4 in cellular systems?

Differentiating between direct and indirect effects of REG4 in cellular systems requires a multi-faceted experimental approach. Temporal analysis provides the first line of discrimination—immediate responses (within minutes to 1-2 hours) following REG4 treatment are more likely direct effects, while changes observed only after several hours typically represent secondary responses mediated by intermediate factors .

Pharmacological inhibition studies using pathway-specific inhibitors can identify dependency relationships. If blocking a specific pathway (e.g., EGFR or Akt inhibitors) prevents a REG4-induced effect, that effect likely requires the inhibited pathway as an intermediary. Combining REG4 treatment with protein synthesis inhibitors (cycloheximide) or transcription inhibitors (actinomycin D) can determine whether new protein synthesis or transcription is required for observed effects—direct effects should persist despite these inhibitors .

Receptor competition assays using blocking antibodies against putative REG4 receptors (CD44, GPR37) or excess soluble receptor fragments can confirm receptor dependency. Receptor knockout or knockdown approaches provide complementary evidence—loss of response in receptor-deficient cells strongly suggests direct receptor-mediated effects .

For signal transduction studies, phosphorylation kinetics analysis comparing REG4 with known direct activators (e.g., EGF for EGFR pathway) can reveal similarities in response profiles indicative of direct activation. Finally, in vitro reconstitution using purified components can provide definitive evidence—if REG4 can activate purified EGFR in a cell-free system, direct interaction is confirmed. These complementary approaches collectively enable robust discrimination between direct and indirect REG4 effects .

What are the emerging questions in REG4 biology that warrant further investigation?

Several critical questions in REG4 biology remain unresolved and warrant focused investigation. The full spectrum of REG4 receptors beyond CD44 and GPR37 remains poorly characterized—systematic screens for additional binding partners using technologies like CRISPR-based genome-wide screens or protein microarrays could identify novel receptors mediating tissue-specific effects .

The mechanistic basis for REG4's dual role in inflammation and cancer requires clarification, particularly how it enhances macrophage polarization to M2 phenotypes while simultaneously promoting tumor progression. The potential role of REG4 in mediating tumor-immune system interactions, especially in the context of immunotherapy response, represents an unexplored frontier .

The functional significance of REG4 glycosylation patterns demands investigation—how specific glycan structures affect receptor binding, signaling outcomes, and protein stability remains largely unknown. The contribution of REG4 to cancer stem cell maintenance and therapy resistance mechanisms requires deeper exploration, particularly the molecular pathways connecting REG4 signaling to stemness factors like SOX2 .

The reciprocal regulatory relationship between REG4 and the microenvironment represents another knowledge gap—how stromal cells, extracellular matrix components, and metabolic conditions modulate REG4 expression and function. Finally, the potential role of REG4 as a liquid biopsy biomarker for early cancer detection or monitoring treatment response deserves rigorous evaluation across multiple cancer types. These questions highlight promising directions for advancing our understanding of REG4 biology .

What novel methodological approaches might advance our understanding of REG4 functions?

Advancing our understanding of REG4 functions requires innovative methodological approaches that overcome current technical limitations. CRISPR-based approaches beyond simple knockouts—including CRISPRa/CRISPRi for fine-tuned expression modulation and base editing for introducing specific mutations in REG4 or its receptors—will provide nuanced insights into structure-function relationships .

Organoid models derived from normal and cancerous tissues represent a significant advancement over traditional cell culture, recapitulating tissue architecture and cellular heterogeneity for studying REG4 in more physiologically relevant contexts. These can be combined with microfluidic organ-on-chip platforms incorporating multiple cell types (epithelial, immune, stromal) to model complex REG4-mediated intercellular communications .

Single-cell technologies applied to REG4 biology—including single-cell RNA-seq, CyTOF, and spatial transcriptomics—can reveal cell-type specific responses and spatial regulation patterns impossible to detect in bulk analyses. For structural insights, cryo-electron microscopy of REG4-receptor complexes can elucidate binding interfaces and conformational changes upon interaction .

In vivo imaging approaches using antibody-based or aptamer-based probes for REG4 can track its distribution and activity in real-time in animal models. Integrative multi-omics approaches combining transcriptomics, proteomics, phospho-proteomics, and metabolomics data will provide systems-level understanding of REG4 signaling networks. Finally, machine learning algorithms applied to large-scale REG4-related datasets can identify novel patterns and generate testable hypotheses about REG4 functions across diverse biological contexts .

These innovative methodological approaches, particularly when used in combination, promise to substantially advance our understanding of REG4 biology in both normal physiology and disease states.