INF2 Antibody

What is INF2 and what structural characteristics should researchers be aware of?

INF2 (inverted formin, FH2 and WH2 domain containing) is a 135.6 kilodalton protein that belongs to the formin family. It is also known by alternative names including C14orf173, C14orf151, CMTDIE, FSGS5, inverted formin-2, and HBEAG-binding protein 2 binding protein C . Unlike conventional formins, INF2 has the unique capacity to both polymerize and depolymerize actin, which makes it particularly important in dynamic cellular processes. When selecting antibodies, researchers should consider which specific domains they wish to target, as different epitopes may reveal distinct functional aspects of INF2.

What applications are INF2 antibodies most commonly validated for?

Based on current research tools, INF2 antibodies have been validated for several critical applications:

Most commercially available antibodies are validated for Western blot applications, with many also suitable for immunoprecipitation studies . When planning experiments, researchers should verify that their chosen antibody has been specifically validated for their intended application.

How should researchers validate INF2 antibody specificity for their experiments?

Validation should follow a multi-step approach:

• Positive and negative controls: Use tissues or cell lines known to express or lack INF2 expression.

• Knockout/knockdown validation: As demonstrated in recent research, comparing staining between parental and INF2 KO cell lines provides definitive validation .

• Peptide competition: Pre-incubation with the immunizing peptide should abolish specific binding.

• Multiple antibody comparison: Use antibodies targeting different epitopes of INF2 to confirm staining patterns.

• Molecular weight verification: INF2 should appear at approximately 135.6 kDa in Western blots.

A recent study examining INF2 in endometrial cancer validated antibody specificity using CRISPR/Cas9-generated INF2 knockout HEC-1B cells before proceeding with IHC analysis of tissue microarrays . This represents the gold standard for antibody validation.

How can researchers detect specific phosphorylation states of INF2?

Recent research has identified that AMPK phosphorylates INF2 at Ser1077, which affects its localization and function in mitochondrial dynamics . To detect this specific phosphorylation:

• Phospho-specific antibodies: Select antibodies specifically targeting phospho-Ser1077 if available.

• Phosphatase treatment controls: Compare samples with and without phosphatase treatment.

• Mutational analysis: Compare wild-type INF2 with phospho-null mutants (e.g., S1077A) and phosphomimetic mutants.

• Kinase inhibitor studies: Use AMPK inhibitors to manipulate phosphorylation state.

• Mass spectrometry validation: For definitive identification of phosphorylation sites.

The phosphorylation status of INF2 significantly affects its subcellular localization and function in mitochondrial fission processes, making this a critical consideration for researchers investigating mitochondrial dynamics .

What methodological approaches are recommended for studying INF2-mediated mitochondrial dynamics?

INF2 plays a crucial role in mitochondrial fission through actin polymerization. To effectively study this:

• Live-cell imaging: Use fluorescently tagged INF2 with mitochondrial markers.

• Super-resolution microscopy: Required to visualize INF2-actin interactions at mitochondrial constriction sites.

• Mitochondrial morphology analysis: Quantify parameters like length, branching, and network connectivity using software such as MiNA or MitoGraph.

• Pharmacological interventions: Use inhibitors like MFI8 (a mitochondrial fusion inhibitor) to distinguish between fusion and fission defects .

• Co-immunoprecipitation: To identify INF2 interactions with mitochondrial fission machinery like DRP1.

Recent studies have demonstrated that INF2 knockout cells show increased mitochondrial length and fusion into tube-like structures with reduced division, confirming INF2's role in controlling mitochondrial dynamics .

How can researchers optimize INF2 co-localization studies with the endoplasmic reticulum (ER)?

As INF2 resides in the ER and mediates ER-mitochondria interactions:

• Fixation optimization: Use fixation methods that preserve ER structure (4% PFA for 10-15 minutes).

• Permeabilization considerations: Mild detergents (0.1% Triton X-100 or 0.1% saponin) are preferable.

• Multi-channel imaging: Simultaneously visualize INF2, ER markers (e.g., calnexin), and mitochondrial markers.

• Z-stack acquisition: Collect multiple focal planes for 3D reconstruction.

• Quantitative co-localization analysis: Use Pearson's or Mander's coefficients to quantify co-localization.

Under energy stress conditions, phosphorylation of INF2 at Ser1077 increases its localization to the ER and enhances recruitment of DRP1 to mitochondria, making co-localization studies particularly informative for understanding mitochondrial fission regulation .

How should researchers approach INF2 expression analysis in cancer tissues?

Recent studies reveal that INF2 expression is significantly upregulated in endometrial cancer and associated with poor prognosis . For comprehensive analysis:

• Tissue microarray (TMA) analysis: Use validated antibodies with proper controls.

• Scoring systems: Implement quantitative scoring of staining intensity and percentage of positive cells.

• Clinical correlation: Correlate expression with tumor stage, grade, and patient outcomes.

• Isoform-specific analysis: Consider potential differential expression of INF2 isoforms.

• Multi-omics integration: Combine protein expression data with mRNA expression and genomic alterations.

What functional assays are recommended for investigating INF2's role in cancer progression?

Based on recent literature:

• Proliferation assays: CCK-8, colony formation, and EdU incorporation assays have demonstrated reduced growth in INF2 KO cancer cells .

• Migration and invasion assays: Transwell and wound healing assays to assess metastatic potential.

• Mitochondrial function assessment: Measure parameters like oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).

• In vivo tumorigenesis: Xenograft models comparing INF2 wild-type and knockout cells.

• Rescue experiments: Reintroduce wild-type or mutant INF2 into knockout cells to confirm phenotypes.

Recent research used CRISPR/Cas9-based genome editing to generate INF2 KO cancer cell lines, which showed significantly reduced growth in vitro, establishing a methodological framework for such studies .

What are the optimal conditions for Western blot detection of INF2?

For reliable Western blot results:

• Sample preparation: Use RIPA buffer supplemented with protease and phosphatase inhibitors.

• Protein loading: 20-50 μg of total protein per lane is typically sufficient.

• Gel percentage: 6-8% gels are optimal for resolving the 135.6 kDa INF2 protein.

• Transfer conditions: Wet transfer for 2 hours at 100V or overnight at 30V at 4°C.

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Incubation at 1:500-1:2000 dilution overnight at 4°C.

• Washing: 3-5 times with TBST for 5-10 minutes each.

• Secondary antibody: HRP-conjugated at 1:5000-1:10000 for 1 hour at room temperature.

Multiple commercially available antibodies have been validated for Western blot detection of INF2, with confirmed specificity through knockout cell validation .

How can researchers optimize immunohistochemical detection of INF2 in tissue sections?

Based on successful IHC protocols:

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

• Endogenous peroxidase quenching: 3% hydrogen peroxide for 10 minutes.

• Blocking: 5-10% normal serum from the same species as the secondary antibody.

• Primary antibody: Optimize concentration (typically 1:100-1:500) with overnight incubation at 4°C.

• Detection system: Polymer-based detection systems generally provide better signal-to-noise ratios.

• Counterstaining: Hematoxylin for nuclear visualization.

• Controls: Include positive control tissues, negative controls (primary antibody omission), and ideally INF2 knockout tissue controls.

Researchers have successfully used IHC analysis with validated INF2 antibodies to demonstrate increased expression in endometrial cancer tissues compared to adjacent normal tissues .

Why might researchers observe multiple bands when probing for INF2 in Western blots?

Multiple bands may appear due to:

• Alternative splicing: INF2 has multiple isoforms that may appear as distinct bands.

• Post-translational modifications: Phosphorylation at sites like Ser1077 can cause mobility shifts .

• Proteolytic degradation: Sample preparation without adequate protease inhibitors.

• Non-specific binding: Insufficient blocking or antibody cross-reactivity.

• Splice variants: Different tissues may express different INF2 variants.

To address this issue, researchers should use multiple antibodies targeting different epitopes and include appropriate controls such as INF2 knockout samples to identify specific bands .

How should researchers interpret differential INF2 expression across cancer cell lines?

Studies have shown variable INF2 expression across endometrial cancer cell lines . For proper interpretation:

• Baseline characterization: Quantify INF2 at both mRNA and protein levels across multiple cell lines.

• Correlation with phenotype: Assess whether expression levels correlate with cellular behaviors like proliferation or migration.

• Isoform analysis: Determine if specific isoforms are preferentially expressed in certain cell lines.

• Genetic background consideration: Consider the genetic context of each cell line.

• Functional validation: Perform knockdown/knockout studies in high-expressing lines and overexpression in low-expressing lines.

Recent research observed abundant INF2 expression in most endometrial cancer cell lines, with exceptions such as SPEC-2 and AN3CA cells, suggesting cell-type-specific regulation .

What methodologies are recommended for investigating INF2-AMPK interactions?

Based on recent findings linking AMPK to INF2 phosphorylation:

• Co-immunoprecipitation: Using antibodies against either INF2 or AMPK.

• In vitro kinase assays: To directly measure AMPK-mediated phosphorylation of INF2.

• Proximity ligation assays: To visualize INF2-AMPK interactions in situ.

• FRET/BRET analysis: For real-time monitoring of protein interactions.

• Pharmacological modulation: Use AMPK activators (e.g., AICAR, metformin) or inhibitors (e.g., Compound C).

• Mutational analysis: Compare interaction with wild-type versus phospho-null (S1077A) INF2 .

Recent research has established that AMPK phosphorylates INF2 at Ser1077 under energy stress conditions, highlighting the importance of studying this regulatory mechanism .

How can researchers design experiments to study the therapeutic potential of targeting INF2 in cancer?

For translational research approaches:

• Small molecule screening: Identify compounds that modulate INF2 activity or expression.

• PROTAC development: Design proteolysis-targeting chimeras to selectively degrade INF2.

• Combinatorial approaches: Test INF2 targeting in combination with standard chemotherapies.

• Patient-derived xenografts: Evaluate effects in models that better recapitulate human tumors.

• Biomarker development: Correlate INF2 expression with treatment response.

• Mitochondrial function targeting: Focus on the role of INF2 in mitochondrial dynamics, which is often dysregulated in cancer.

Given the correlation between high INF2 expression and poor prognosis in endometrial cancer, targeting INF2 or its downstream pathways represents a promising therapeutic avenue .