Recombinant Mouse Protein LDOC1 (Ldoc1)

What is the general structural organization of LDOC1 protein?

LDOC1 protein contains a leucine zipper domain, which is crucial for protein-protein interactions through dimerization. The protein also contains a proline-rich region that may mediate interactions with other cellular proteins. Studies indicate that LDOC1 can physically interact with several proteins including MZF1, PEG10, ABLIM1, ZNF253, NOD2, CEBPE, ZBTB16, HGS, and GNL3L, suggesting its involvement in multiple cellular processes .

What expression systems are commonly used for recombinant mouse LDOC1 production?

Several expression systems have been utilized for recombinant LDOC1 production, each with distinct advantages:

• HEK-293 Cells: Mammalian expression system that provides proper post-translational modifications and folding, yielding proteins with >90% purity as determined by Bis-Tris PAGE, anti-tag ELISA, Western Blot, and analytical SEC .

• E. coli: Bacterial expression system commonly used for high-yield production of recombinant proteins, typically achieving >95% purity for LDOC1 .

• Cell-free protein synthesis (CFPS): This system allows for rapid protein production without cellular constraints, typically achieving 70-80% purity as determined by SDS-PAGE, Western Blot, and analytical SEC .

The choice of expression system should be determined by the specific experimental requirements, including protein folding complexity, post-translational modification needs, and application purpose.

How does LDOC1 expression differ between normal and cancerous tissues?

LDOC1 exhibits significantly different expression patterns between normal and cancerous tissues. Analysis from GEPIA and UALCAN databases has demonstrated that LDOC1 is highly expressed in normal tissue samples but shows markedly reduced expression in tumor tissues (p < 0.05) . This differential expression has been validated through multiple experimental approaches:

• Real-time polymerase chain reaction (RT-PCR)

• Western blot analysis

• Immunohistochemistry (IHC)

In hepatocellular carcinoma (HCC) specifically, LDOC1 expression negatively correlates with tumor grade progression, with lower expression observed in more advanced tumors . This pattern suggests that LDOC1 downregulation may play a role in cancer development and progression.

What molecular techniques are most effective for quantifying LDOC1 expression?

Based on research protocols, the following techniques have proven effective for LDOC1 quantification:

• RT-PCR using specific primers:

  • Forward: 5'-ATGAAGGTGGCATTCCTAATCA-3'

  • Reverse: 5'-AATCATCCTCCTCTTCTTCGTC-3'

• Western blot analysis using specific antibodies:

  • For protein detection, antibodies such as cat. no. 10113-2-AP (Proteintech) have been successfully employed .

• Immunohistochemistry (IHC):

  • Using antibodies such as cat. no. PA5-50438 (Thermo Fisher Scientific) .

  • Quantification through histochemistry score (H-score) to determine positive intensity.

• Flow cytometry:

  • For analysis of cellular effects following LDOC1 manipulation.

These complementary approaches provide robust validation of LDOC1 expression at both RNA and protein levels.

What are the recommended cell lines for studying LDOC1 function in cancer research?

Based on published research, the following cell lines have been successfully used for LDOC1 functional studies:

• Huh7: Human hepatocellular carcinoma cell line that has shown significant responses to LDOC1 overexpression, including altered cell cycle distribution, reduced proliferation, and increased apoptosis .

• Hep3B: Another human hepatocellular carcinoma cell line used to validate findings from Huh7 cells, demonstrating consistent responses to LDOC1 manipulation .

These cell lines provide reliable models for studying LDOC1's effects on cancer-related cellular processes. When selecting a cell line, researchers should consider:

• Baseline LDOC1 expression levels

• Cancer type relevance

• Transfection efficiency

• Growth characteristics

How does LDOC1 influence cell cycle progression and apoptosis?

LDOC1 has demonstrated significant effects on cell cycle distribution and apoptosis:

• Cell Cycle Effects:

  • In Huh7 cells, LDOC1 overexpression increases the proportion of cells in G1 and G2 phases

  • In Hep3B cells, LDOC1 overexpression primarily increases the G2 phase population

• Apoptotic Effects:

  • LDOC1 overexpression significantly increases the rate of apoptosis compared to control groups (p < 0.05)

  • This can be measured using Annexin V-Allophycocyanin (APC)/7-Amino-Actinomycin D (7-AAD) staining and flow cytometry analysis

These effects contribute to LDOC1's tumor-suppressive properties, as they collectively reduce cancer cell proliferation and survival.

What signaling pathways does LDOC1 interact with to mediate its effects?

Research indicates that LDOC1 influences several signaling pathways, most notably the AKT/mTOR pathway:

• AKT/mTOR Pathway:

  • LDOC1 upregulation reduces the phosphorylation ratios of both AKT and mTOR

  • Specifically, p-AKT/AKT and p-mTOR/mTOR ratios decrease following LDOC1 overexpression

  • This suggests that LDOC1 exerts its tumor-suppressive effects partly through inactivation of the AKT/mTOR pathway

• Protein-Protein Interactions:

  • LDOC1 physically interacts with multiple proteins including MZF1, PEG10, ABLIM1, ZNF253, NOD2, CEBPE, ZBTB16, HGS, and GNL3L

  • Protein-protein interaction analysis also shows LDOC1 interaction with PEG10, GNL3L, MZF1, PNMA5, RGAG1, ZCCHC16, TPRKB, CLIC1, SPANXD, and FTH1

These interactions suggest that LDOC1 may influence cellular processes through multiple mechanisms beyond AKT/mTOR signaling.

What is the optimal protocol for establishing LDOC1 overexpression models?

Based on successful research methodologies, the following protocol is recommended for LDOC1 overexpression:

• Recombinant Lentiviral Vector Construction:

  • Design and synthesize recombinant lentiviral vectors containing LDOC1 (Lv-LDOC1) and empty vector controls (Lv-NC)

  • Include appropriate selection markers (e.g., puromycin resistance) in the plasmid design

• Cell Transfection:

  • Culture target cells (e.g., Huh7 and Hep3B) in appropriate medium with 10% fetal bovine serum

  • Add 5 μg/ml polybrene to enhance transfection efficiency

  • Transfect cells with Lv-NC or Lv-LDOC1 vectors following standard transfection protocols

• Stable Cell Line Selection:

  • After 48 hours of transfection, add 5 μg/ml puromycin to the complete medium

  • Maintain selection pressure for 3-5 weeks at 37°C

  • Regularly change medium containing selection antibiotic

• Validation of Overexpression:

  • Confirm LDOC1 upregulation using RT-PCR and Western blot

  • Compare expression levels between Lv-LDOC1 and Lv-NC groups

This protocol has been validated to produce stable LDOC1 overexpression for subsequent functional studies.

What methods should be used to assess LDOC1's impact on cell migration?

To evaluate LDOC1's effects on cell migration, the following methods have proven effective:

• Wound-Healing Assay (Scratch Assay):

  • Seed transfected cells in six-well plates and allow them to reach confluence

  • Create a "wound" by scratching the cell monolayer with a sterile pipette tip

  • Measure migration distances at 0 and 48 hours

  • Calculate the percentage of wound closure

• Transwell Migration Assay:

  • Place transfected cells in the upper chamber of a transwell insert without matrigel

  • Add chemoattractant to the lower chamber

  • After appropriate incubation time, fix and stain migrated cells

  • Quantify migration by counting cells that traversed the membrane

• Real-Time Cell Analysis (RTCA):

  • Use specialized equipment to monitor cell migration in real-time

  • Provides continuous data on migration dynamics rather than endpoint measurements

Research has shown that LDOC1 overexpression significantly decreases cell migration compared to control groups (p < 0.05), which can be accurately quantified using these methods .

How does LDOC1 expression correlate with patient prognosis in cancer?

LDOC1 expression levels have shown significant correlations with patient outcomes across several survival metrics:

These correlations have been established through Kaplan-Meier survival analysis using LDOC1-23641 as the unique RNA-Seq ID. Stratified analyses based on tumor stages, AJCC_T classification, alcohol consumption, and hepatitis virus infection further support LDOC1's prognostic value .

What is the potential of LDOC1 as a biomarker for cancer diagnosis or prognosis?

Research supports LDOC1's potential as a biomarker:

• Expression Pattern:

  • Consistent downregulation in tumor tissues compared to normal tissues

  • Negative correlation with tumor grade progression

• Prognostic Value:

  • Significant associations with multiple survival metrics (OS, PFS, RFS, DSS)

  • Performance in stratified analyses across different patient subgroups

• Functional Relevance:

  • Demonstrated effects on hallmark cancer processes (proliferation, apoptosis, migration)

  • Involvement in key signaling pathways (AKT/mTOR)

These characteristics suggest that LDOC1 could serve as a valuable prognostic biomarker, potentially guiding treatment decisions and risk stratification for cancer patients, particularly those with hepatocellular carcinoma.

What are the optimal storage conditions for recombinant mouse LDOC1 protein?

To maintain protein stability and activity, recombinant mouse LDOC1 protein should be stored according to the following recommendations:

• Temperature: Store at -80°C for long-term preservation

• Physical State: Maintain in liquid form

• Avoid: Repeated freeze-thaw cycles that can denature the protein

Properly stored LDOC1 protein typically maintains activity for 12 months from the date of receipt . For working solutions, minimize the number of freeze-thaw cycles by preparing single-use aliquots before storage.

What controls should be included in LDOC1 functional studies?

To ensure experimental validity when studying LDOC1 function, incorporate the following controls:

• Empty Vector Control:

  • Use cells transfected with empty vector (e.g., Lv-NC) as the primary control for overexpression studies

  • This controls for any effects of the transfection process itself

• Wild-Type Cells:

  • Include untransfected cells to establish baseline parameters

• Positive Controls:

  • For apoptosis studies: Include cells treated with a known apoptosis inducer

  • For proliferation studies: Include rapidly proliferating control cell populations

• Functional Validation:

  • Confirm LDOC1 expression changes at both mRNA and protein levels

  • Use RT-PCR and Western blot to validate expression in experimental and control groups

• Multiple Cell Lines:

  • Validate key findings in at least two cell lines (e.g., both Huh7 and Hep3B for HCC studies)

These controls help ensure that observed effects are specifically attributable to LDOC1 rather than experimental artifacts.