ERGIC3 Antibody

What is ERGIC3 and what is its cellular localization?

ERGIC3 (Endoplasmic Reticulum-Golgi Intermediate Compartment protein 3) is located in the cis face of the Golgi apparatus and the vesicular tubular structures between the transitional endoplasmic reticulum (ER) and cis-Golgi. This protein significantly influences cell growth, can trigger ER stress-induced cell death, and plays a role in the invasion and metastasis of hepatocellular carcinomas (HCC) . ERGIC3 forms a heteromeric complex with ERGIC2, and these proteins cycle between the ER and Golgi, functioning as cargo receptors in both anterograde and retrograde protein trafficking pathways .

What applications are validated for ERGIC3 antibodies?

ERGIC3 antibodies are validated for multiple experimental applications according to technical documentation. The most commonly validated applications include:

These applications require optimization in each specific experimental system to obtain optimal results .

What is the typical molecular weight observed for ERGIC3 in Western blots?

While the calculated molecular weight of ERGIC3 is 43 kDa (383 amino acids), the observed molecular weight in Western blot analysis is approximately 50 kDa . This discrepancy is likely due to post-translational modifications of the protein. When performing Western blots, researchers should expect to detect a band at approximately 50 kDa when using anti-ERGIC3 antibodies .

What are the optimal storage conditions for ERGIC3 antibodies?

For maintaining antibody stability and activity, ERGIC3 antibodies should be stored at -20°C where they remain stable for one year after shipment. The standard storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Importantly, aliquoting is generally unnecessary for -20°C storage, which simplifies handling procedures. Some preparations may contain 0.1% BSA in smaller 20μl sizes .

What antigen retrieval methods are recommended for IHC applications?

For optimal results in immunohistochemistry applications using ERGIC3 antibodies, the suggested antigen retrieval method employs TE buffer at pH 9.0. Alternatively, antigen retrieval may be performed with citrate buffer at pH 6.0. This flexibility in retrieval methods allows researchers to adapt protocols to specific tissue types or fixation conditions .

How can researchers validate the specificity of ERGIC3 antibodies?

Antibody specificity can be validated through multiple complementary approaches. The monoclonal antibody 6-C4 was tested by ELISA against the ERGIC3 peptide and native protein from NSCLC cells, showing specific reactivity. Western blot analysis demonstrated a single band at approximately 50 kDa, consistent with the expected molecular weight of ERGIC3. Additionally, immunofluorescence staining localized the signal around the Golgi apparatus and ER, matching the known subcellular localization of ERGIC3. Immunohistochemistry staining of NSCLC and HCC tissues further confirmed antibody specificity .

How can researchers study ERGIC3-dependent protein trafficking?

To investigate ERGIC3's role in protein trafficking, researchers can employ multiple methodological approaches. Evidence indicates that α1-antitrypsin and haptoglobin specifically bind to ERGIC3 and function as its cargo proteins. Experimental designs should include:

• Secretion assays following ERGIC3 depletion (showing decreased secretion of cargo proteins)

• Co-immunoprecipitation studies to identify protein-protein interactions

• Transfection with secreted protein expression plasmids followed by collection and analysis of conditioned medium

• TCA precipitation of secreted proteins followed by Western blot analysis

These methods have demonstrated that ERGIC3 depletion significantly decreases secretion of α1-antitrypsin and haptoglobin, confirming ERGIC3's role as a cargo receptor .

What methods are effective for studying ERGIC3 ubiquitination?

ERGIC3 ubiquitination can be studied using both in vitro and in vivo approaches:

For in vitro ubiquitination assays:

• Incubate purified ERGIC3-HA with 100 ng HA-Uba1 (E1), 250 ng UbcH5c (E2), 1 μg ubiquitin, and 5 mM ATP

• Include or exclude purified FLAG-MARCH2 WT or FLAG-MARCH2 C64,67S (inactive mutant)

• Perform the reaction in buffer containing 40 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, and 1 mM DTT for 2 hours

• Analyze by immunoblotting with anti-HA antibody

For in vivo ubiquitination assays:

• Transfect cells with FLAG-MARCH2 WT or FLAG-MARCH2 C64,67S

• Treat with MG132 (25 μM) for 6 hours to inhibit proteasomal degradation

• Lyse cells in RIPA buffer containing 1% SDS and 10 mM N-ethylmaleimide

• Immunoprecipitate endogenous ERGIC3 and analyze by Western blotting

How do researchers identify specific ubiquitination sites on ERGIC3?

Research has identified lysine residues at positions 6 and 8 of ERGIC3 as the major sites of MARCH2-mediated ubiquitination. To identify these sites, researchers employed site-directed mutagenesis to generate ERGIC3 variants with lysine-to-arginine substitutions at these positions. Subsequent ubiquitination assays demonstrated that MARCH2 could not significantly decrease the levels of ERGIC3 variants with K6R and K8R substitutions, confirming these residues as the primary ubiquitination targets .

How is ERGIC3 expression altered in cancer tissues?

ERGIC3 shows distinctive expression patterns across different tissue types:

• In normal human tissues: ERGIC3 expression is limited to specific epithelial cells including hepatocytes, gastrointestinal epithelium, pancreatic ducts and acini, kidney tubules, and mammary epithelial cells, while most normal human tissues show negative staining.

• In cancerous tissues: Almost all carcinomas originating from epithelial cells show positive staining for ERGIC3, while all sarcomas tested were negative. This differential expression pattern suggests ERGIC3 may serve as a potential biomarker for carcinomas .

ERGIC3 was initially identified as a novel lung cancer-related gene through screening of differentially expressed gene libraries, suggesting its potential role in cancer biology .

What methodologies are used to study miRNA regulation of ERGIC3 expression?

Research has identified that ERGIC3 is regulated by miR-203a. To study this regulatory relationship, researchers employ several complementary approaches:

• Bioinformatics prediction using algorithms like RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid) and miRecords (http://mirecords.biolead.org/prediction_query.php)

• Expression analysis using qRT-PCR:

  • Total RNA extraction

  • cDNA preparation using miScript II RT Kit with 5× miScript HiSpec Buffer

  • qRT-PCR using miScript SYBR Green PCR Kit with miScript Universal Primer

  • U6 as internal control for normalization

• Validation experiments to confirm direct interaction between miR-203a and ERGIC3 mRNA

What is the potential of ERGIC3 as a biomarker for specific cancer types?

ERGIC3 shows promise as a potential biomarker, particularly for non-small cell lung cancer (NSCLC). The high sensitivity and specificity of ERGIC3 expression in NSCLC, combined with its role in cancer development and progression, suggest significant biomarker potential. The development of monoclonal antibodies like 6-C4 that specifically recognize ERGIC3 provides valuable tools for detecting this protein in clinical samples through techniques like immunohistochemistry .

How does ERGIC3 interact with other ERGIC family proteins?

ERGIC3 displays distinct interaction patterns with other ERGIC family members. Research demonstrates that ERGIC3 can bind both to itself (self-association) and to ERGIC2, forming heteromeric complexes. Interestingly, ERGIC2 is unable to interact with itself, suggesting a specific structural arrangement in these complexes. This interaction pattern indicates that ERGIC3 may serve as a central component in organizing functional complexes within the early secretory pathway .

What is the role of MARCH2 in regulating ERGIC3 function?

The E3 ubiquitin ligase membrane-associated ring-CH-type finger 2 (MARCH2) plays a critical regulatory role in ERGIC3 function through the following mechanisms:

• MARCH2 directs the ubiquitination of ERGIC3, primarily at lysine residues 6 and 8

• This ubiquitination marks ERGIC3 for subsequent degradation

• MARCH2 depletion increases endogenous ERGIC3 levels

• MARCH2 reduces secretion of ERGIC3-dependent cargo proteins (α1-antitrypsin and haptoglobin)

• Expression of ubiquitination-resistant ERGIC3 variants can restore trafficking of these secretory proteins

These findings demonstrate that MARCH2-mediated ERGIC3 ubiquitination represents a key regulatory mechanism in the early secretory pathway .

What experimental approaches demonstrate the cargo receptor function of ERGIC3?

The cargo receptor function of ERGIC3 has been demonstrated through several experimental approaches:

• Binding assays showing that α1-antitrypsin and haptoglobin specifically bind to ERGIC3

• Secretion analysis following ERGIC3 depletion, showing decreased secretion of these cargo proteins

• Rescue experiments using ERGIC3 variants resistant to MARCH2-mediated ubiquitination, which restore secretion of these proteins

• Comparative analysis between control and ERGIC3-depleted cells to quantify differences in cargo protein trafficking

These methodological approaches collectively provide strong evidence for ERGIC3's function as a cargo receptor in the early secretory pathway .

What controls should be included when studying ERGIC3 ubiquitination?

When investigating ERGIC3 ubiquitination, researchers should include the following controls:

• Negative controls:

  • Omission of E1, E2, or E3 enzymes in in vitro assays

  • Use of catalytically inactive MARCH2 C64,67S mutant (with mutations in the RING domain)

  • Non-specific IgG control for immunoprecipitation experiments

• Positive controls:

  • Known ubiquitination substrates of MARCH2

  • Wild-type ERGIC3 in parallel with lysine mutants

• Experimental validation:

  • Use of proteasome inhibitors (MG132, 25 μM for 6 hours) to accumulate ubiquitinated proteins

  • Comparison of wild-type ERGIC3 with K6R/K8R mutants resistant to ubiquitination

How can researchers optimize secretion assays for ERGIC3 cargo proteins?

For optimal analysis of secreted proteins dependent on ERGIC3, researchers should:

• Transiently transfect cells with secreted protein expression plasmids

• After 24 hours, wash cells twice with PBS and incubate in serum-free DMEM for 12 hours

• Collect conditioned medium by centrifugation at 2,000 × g for 5 minutes

• Concentrate proteins using trichloroacetic acid (TCA, 25%) at 4°C for 30 minutes

• Wash precipitated proteins with cold acetone and dry at 50°C for 5 minutes

• Dissolve dried samples in SDS sample buffer and analyze by Western blotting

This protocol allows for effective concentration and detection of secreted proteins, enabling quantitative analysis of ERGIC3's impact on protein secretion .

What are the critical parameters for immunofluorescence localization of ERGIC3?

For accurate immunofluorescence localization of ERGIC3:

• Use validated antibodies at appropriate dilutions (1:50-1:500 for IF/ICC applications)

• Include proper controls:

  • Primary antibody omission control

  • Isotype control

  • Known positive samples

• Co-staining recommendations:

  • Use ERGIC53 as a marker for the ER-Golgi intermediate compartment

  • Consider co-staining with Golgi markers to confirm the expected peri-Golgi localization

The immunofluorescence staining pattern of ERGIC3 should show localization around the Golgi apparatus and endoplasmic reticulum, consistent with its known subcellular distribution .