INSIG1 Antibody

What is INSIG1 and what is its primary function in cellular biology?

INSIG1 (Insulin-induced gene 1) is an endoplasmic reticulum (ER)-resident oxysterol-binding protein with a canonical length of 277 amino acids and a molecular weight of approximately 30 kDa in humans. It belongs to the INSIG family of proteins and functions primarily as a regulator of cholesterol synthesis. INSIG1 mediates feedback control of cholesterol metabolism through two major mechanisms: controlling the endoplasmic reticulum to Golgi transport of SCAP (SREBP cleavage-activating protein) and facilitating the degradation of HMG-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol biosynthesis . Recent research has also revealed an unexpected role for INSIG1 in viral immunity, specifically in inhibiting HIV-1 production through the degradation of the HIV-1 Gag protein, identifying INSIG1 as a potential sentinel protein responsive to viral replication .

What are the expression patterns and subcellular localization of INSIG1?

INSIG1 is expressed across all tested human tissues, with the highest expression levels detected in the liver, consistent with its prominent role in cholesterol metabolism . At the subcellular level, INSIG1 is predominantly localized to the endoplasmic reticulum (ER) membrane, where it interacts with other cholesterol regulatory proteins such as SCAP and HMGCR . This strategic localization allows INSIG1 to function effectively in monitoring and regulating cellular sterol levels through protein-protein interactions. Additionally, research has demonstrated that INSIG1 can be found at intracellular membrane sites including the ER and endosomes, which is particularly relevant for its interaction with HIV-1 Gag during viral infection and replication processes . The proper subcellular localization of INSIG1 is critical for both its canonical sterol-regulatory functions and its newly discovered role in antiviral immunity.

What post-translational modifications affect INSIG1 function?

INSIG1 undergoes several important post-translational modifications that regulate its stability and function. Two primary modifications documented in the literature include ubiquitination and phosphorylation . Ubiquitination of INSIG1 plays a critical role in protein turnover and in its ability to target other proteins (such as HMGCR) for degradation. The phosphorylation status of INSIG1 may modulate its interactions with partner proteins and influence its stability in the ER membrane. These modifications are dynamic and responsive to cellular conditions, particularly sterol levels, allowing INSIG1 to function as an effective sensor and regulator in cholesterol homeostasis. When studying INSIG1 with antibodies, researchers should consider how these post-translational modifications might affect epitope accessibility and antibody binding, especially when working with phospho-specific or modification-specific antibodies .

What are the known isoforms of INSIG1 and how do they differ?

INSIG1 exists in at least two distinct isoforms resulting from alternative splicing of the INSIG1 gene . These isoforms maintain the core functional domains but may exhibit subtle differences in regulatory regions that affect their stability, localization, or interaction capabilities. The canonical isoform consists of 277 amino acids with a molecular weight of approximately 30 kDa, although Western blot analysis using anti-INSIG1 antibodies frequently reveals bands at both 28-31 kDa and 66-70 kDa, suggesting potential dimerization, complex formation, or additional post-translational modifications . When designing experiments involving INSIG1 detection, researchers should consider which isoforms their antibodies recognize and whether their experimental question requires isoform-specific analysis. The functional differences between these isoforms remain an active area of research, particularly in understanding tissue-specific roles and differential responses to metabolic stimuli or viral infection conditions .

How does INSIG1 inhibit HIV-1 production through Gag protein degradation?

INSIG1 exhibits a novel antiviral function by inhibiting HIV-1 production through the targeted degradation of the HIV-1 Gag protein. This mechanism operates distinctly from INSIG1's canonical role in sterol regulation. Unlike the degradation of HMGCR which is mediated by the E3 ubiquitin ligase AMFR through a proteasome-dependent pathway, INSIG1 coordinates with a different E3 ligase called TRC8 (also known as RNF139, Ring Finger Protein 139) to promote Gag degradation specifically through the lysosomal pathway . This process occurs at intracellular membrane sites such as the endoplasmic reticulum and endosomes where both INSIG1 and Gag proteins localize. Experimental evidence supporting this mechanism comes from both gain-of-function and loss-of-function approaches. Overexpression of INSIG1 in both 293T and Jurkat cells significantly inhibited the production of HIV-1 pseudoviruses (both Env and VSV-G enveloped) by reducing intracellular Pr55 Gag levels . Conversely, CRISPR/Cas9-mediated knockout of INSIG1 in these cell lines increased HIV-1 production and was associated with higher accumulation of Gag protein . Importantly, reintroduction of INSIG1 in knockout cells restored the inhibitory effect on HIV-1 production, confirming the specificity of this mechanism. This INSIG1-mediated restriction of HIV-1 appears to be cholesterol-independent, as cellular cholesterol content was not significantly altered during these processes .

What is the relationship between INSIG1 and TRC8 in mediating protein degradation?

The functional relationship between INSIG1 and the E3 ubiquitin ligase TRC8 (Translocation in Renal Carcinoma Chromosome 8, also known as RNF139) represents a critical mechanism in both cellular protein quality control and antiviral defense. Research indicates that INSIG1 forms a complex with TRC8 to target specific proteins for degradation through the lysosomal pathway, as demonstrated in the case of HIV-1 Gag protein degradation . This partnership differs significantly from INSIG1's better-characterized interaction with AMFR (another E3 ligase) that drives proteasomal degradation of HMGCR in sterol metabolism. The INSIG1-TRC8 complex appears to monitor enveloped virion assembly rather than simply recognizing individual viral proteins, as evidenced by the observation that INSIG1 responds to virion generation following co-expression of Gag-pol and Env, but not Gag-pol alone . This sophisticated monitoring system suggests that INSIG1 may act as a cellular sentinel that recognizes patterns associated with viral assembly at membrane interfaces. The molecular details of how INSIG1 recruits TRC8 specifically to viral proteins while sparing host proteins remain to be fully elucidated and represent an important area for future investigation using co-immunoprecipitation, proximity labeling, and structural biology approaches with carefully validated INSIG1 antibodies .

What are the optimal applications for different INSIG1 antibodies and how should they be validated?

Different INSIG1 antibodies demonstrate varying efficacy across experimental applications, requiring careful selection and validation. For Western blot applications, both rabbit polyclonal (e.g., 22115-1-AP) and mouse monoclonal antibodies against INSIG1 have demonstrated efficacy, with recommended dilutions ranging from 1:500 to 1:2000 . For immunohistochemistry applications, the 22115-1-AP antibody can be used at dilutions of 1:20 to 1:200, with specific antigen retrieval protocols using either TE buffer (pH 9.0) or citrate buffer (pH 6.0) depending on the tissue sample . For immunofluorescence applications, validation data shows effectiveness in multiple human cell lines, providing spatial information about INSIG1 localization.

A comprehensive validation protocol for INSIG1 antibodies should include:

• Western blot analysis using positive control lysates from cells known to express INSIG1 (A549, HepG2, SGC-7901, MCF-7 cells)

• Knockout/knockdown controls to verify specificity (as demonstrated in CRISPR/Cas9 INSIG1 knockout cell lines)

• Peptide competition assays to confirm epitope specificity

• Cross-reactivity testing in multiple species if cross-species applications are intended

• Application-specific validation for specialized techniques like ChIP, flow cytometry, or IP

Researchers should titrate antibodies in each experimental system to determine optimal working concentrations, as sensitivity can be sample-dependent .

What are the recommended protocols for detecting INSIG1 in different sample types?

Detection of INSIG1 across different sample types requires optimized protocols tailored to the specific preparation method and application. The following methodological approaches are recommended based on validated experimental data:

For Western Blot detection in cell lysates:

• Prepare lysates using RIPA buffer containing protease inhibitors

• Load 20-40 μg of total protein per lane

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF membrane (recommended over nitrocellulose for hydrophobic proteins)

• Block with 5% non-fat milk in TBST for 1 hour

• Incubate with primary anti-INSIG1 antibody (1:500-1:2000 dilution) overnight at 4°C

• Look for bands at both 28-31 kDa (monomeric form) and 66-70 kDa (potential dimer/complex)

For Immunohistochemistry in tissue sections:

• Use FFPE sections (4-6 μm thickness)

• Perform antigen retrieval with TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative)

• Block endogenous peroxidase with 3% H₂O₂

• Block non-specific binding with serum-free protein block

• Incubate with primary anti-INSIG1 antibody at 1:20-1:200 dilution

• Human liver and kidney tissues serve as positive controls

For Immunofluorescence in cultured cells:

• Grow cells on coverslips to 70-80% confluence

• Fix with 4% paraformaldehyde for 15 minutes

• Permeabilize with 0.1% Triton X-100 for 10 minutes

• Block with 1% BSA in PBS for 30 minutes

• Incubate with anti-INSIG1 antibody overnight at 4°C

• Counter-stain with ER markers to confirm subcellular localization

Each protocol should be optimized for the specific cell type or tissue being analyzed, with careful attention to negative and positive controls .

How can INSIG1 antibodies be used to study protein-protein interactions?

To effectively investigate INSIG1's protein-protein interactions, researchers can employ several antibody-dependent methodologies, each with specific technical considerations:

Co-Immunoprecipitation (Co-IP):

• Prepare cell lysates under mild, non-denaturing conditions (NP-40 or Triton X-100 based buffers)

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Immunoprecipitate with anti-INSIG1 antibody (preferably pre-validated for IP applications)

• Analyze precipitated complexes by Western blot, probing for suspected interaction partners (e.g., TRC8, SCAP, HMGCR)

• Perform reciprocal IP with antibodies against interaction partners to confirm bidirectional pull-down

Proximity Ligation Assay (PLA):

• Fix and permeabilize cells as for standard immunofluorescence

• Incubate with primary antibodies targeting INSIG1 and its potential interaction partner (must be from different species)

• Apply species-specific PLA probes, ligase, and polymerase according to manufacturer protocols

• Quantify fluorescent dots representing in situ protein interactions within 40 nm proximity

FRET/BRET Analyses:

• Generate expression constructs for INSIG1 and interaction partners fused to appropriate fluorophores

• Co-transfect into target cells (HEK293T, HepG2, or Jurkat cells depending on research question)

• Measure energy transfer efficiency using spectrophotometric or microscopy-based approaches

• Validate interactions with antibody-based techniques

When studying INSIG1's interaction with TRC8 in the context of HIV-1 Gag degradation, researchers should consider membrane compartmentalization, as these interactions occur at the ER and endosomal membranes . Detergent selection is critical—digitonin or low concentrations of NP-40 help preserve membrane-associated complexes. Crosslinking approaches (DSP, formaldehyde) may stabilize transient interactions before immunoprecipitation with anti-INSIG1 antibodies . These methods should be complemented with functional assays (e.g., degradation assays) to establish biological relevance of identified interactions.

How can researchers address non-specific binding issues with INSIG1 antibodies?

Non-specific binding presents a common challenge when working with INSIG1 antibodies, potentially leading to misleading results and interpretations. To address these issues effectively, researchers should implement a systematic troubleshooting approach:

For Western Blot applications:

• Compare observed band patterns with expected molecular weights: INSIG1 should appear primarily at 28-31 kDa, with additional bands potentially at 66-70 kDa representing dimers or complexes

• Implement stringent blocking protocols using 5% BSA rather than milk for phospho-specific detection

• Increase washing stringency with higher salt TBST (0.1% to 0.3% Tween-20)

• Titrate antibody concentration using dilution series (1:500 to 1:2000) to identify optimal signal-to-noise ratio

• Include knockout/knockdown controls alongside wild-type samples to identify non-specific bands

• For particularly problematic samples, consider pre-adsorption of antibodies with cell lysates from INSIG1 knockout cells

For Immunohistochemistry/Immunofluorescence:

• Optimize antigen retrieval conditions (compare TE buffer pH 9.0 and citrate buffer pH 6.0)

• Implement dual blocking strategy with serum followed by protein block

• Include peptide competition controls where primary antibody is pre-incubated with immunizing peptide

• Counterstain with established subcellular markers (ER markers like calnexin) to confirm specificity of localization pattern

• Compare staining patterns across multiple validated INSIG1 antibodies targeting different epitopes

Advanced troubleshooting for persistent issues:

• Consider membrane protein extraction optimization (avoid harsh detergents that might disrupt epitopes)

• Implement gradient gels for better separation of closely migrating proteins

• For tissues with high lipid content (e.g., liver), modify extraction protocols to reduce lipid interference

By methodically implementing these approaches and carefully documenting optimization steps, researchers can significantly improve specificity when detecting INSIG1 in various experimental contexts .

How should researchers interpret apparent discrepancies in INSIG1 molecular weight on Western blots?

Researchers frequently encounter apparent discrepancies between the calculated molecular weight of INSIG1 (30 kDa) and its observed migration pattern on Western blots, which can include bands at 28-31 kDa as well as higher molecular weight bands at 66-70 kDa . These variations require careful interpretation based on several biological and technical factors.

The 28-31 kDa band range represents the monomeric form of INSIG1, with slight variations that may result from:

• Post-translational modifications, particularly phosphorylation and ubiquitination, which can alter the migration pattern

• Detection of different isoforms resulting from alternative splicing

• Sample preparation conditions affecting protein denaturation (complete vs. partial)

• Different gel systems and running conditions affecting apparent molecular weight

The higher molecular weight bands (66-70 kDa) frequently observed can represent:

• Stable dimers of INSIG1 that resist denaturing conditions

• INSIG1 in complex with smaller interaction partners

• Ubiquitinated forms of INSIG1, particularly relevant given its role in protein degradation pathways

• Cross-reactivity with related proteins (less likely when using validated antibodies)

To properly interpret these patterns, researchers should:

• Include appropriate positive control lysates from cells known to express INSIG1 (A549, HepG2, SGC-7901, MCF-7)

• Compare results from multiple antibodies targeting different epitopes

• Perform validation with INSIG1 knockout/knockdown samples to confirm band specificity

• Consider using gradient gels for better resolution of closely migrating species

• Complement Western blot findings with mass spectrometry analysis for definitive identification

When studying INSIG1's role in HIV-1 Gag degradation, researchers should be particularly attentive to changes in the relative intensities of these bands, as shifts between monomeric and complexed forms may reflect functional states relevant to the TRC8-mediated degradation pathway .

What controls are essential when studying INSIG1's role in HIV-1 replication?

When investigating INSIG1's role in HIV-1 replication, a comprehensive set of controls is essential to ensure experimental validity and accurate interpretation of results. Based on published research methodologies, the following controls should be implemented:

Genetic Controls:

• INSIG1 knockout cells: CRISPR/Cas9-generated INSIG1 knockout cell lines provide critical negative controls for antibody specificity and functional studies

• Rescue experiments: Reintroduction of INSIG1 with native promoter and synonymous encoding sequence in knockout cells confirms phenotype specificity

• INSIG2 controls: To distinguish INSIG1-specific effects from general INSIG family functions, parallel experiments with INSIG2 manipulation

• Off-target controls: Additional cell lines with knockout of unrelated genes to control for CRISPR/Cas9 effects

Experimental Controls for HIV-1 Studies:

• Envelope variation: Compare results between Env and VSV-G enveloped pseudovirus to distinguish assembly vs. entry effects

• Separate expression: Individual expression of HIV-1 components (Gag-pol vs. Env) to isolate stage-specific effects

• Multiple HIV-1 strains: Test lab-adapted and primary isolates to ensure breadth of effect

• Cell type controls: Validate findings in both model cell lines (293T) and natural HIV-1 target cells (Jurkat)

• Time-course analysis: Monitor INSIG1 expression and HIV-1 production over time to establish temporal relationships

Pathway Validation Controls:

• Lysosome inhibitors: Bafilomycin A1 or chloroquine treatment to confirm lysosomal degradation pathway involvement

• TRC8 knockdown/overexpression: Manipulate proposed E3 ligase partner to confirm mechanism

• Ubiquitination assays: Detect ubiquitinated Gag species in presence/absence of INSIG1

• Cholesterol manipulation: Alter cellular cholesterol to test independence from INSIG1's canonical function

Technical Controls for Antibody-Based Detection:

• Multiple anti-INSIG1 antibodies: Use antibodies targeting different epitopes to confirm consistent results

• Loading controls: Appropriate housekeeping proteins to normalize expression levels

• Subcellular fraction controls: Markers for ER, endosomal, and other compartments when assessing localization