INPP5F Antibody

What is INPP5F and what are its known functions in cellular processes?

INPP5F is a polyphosphoinositide phosphatase that degrades both phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) by removing the 5' phosphate from the inositol ring . It functions as an important endogenous modulator of cardiac myocyte size and stress response . In different cancer contexts, INPP5F demonstrates variable functions - acting as a tumor suppressor in gliomas by inhibiting STAT3 signaling , while displaying oncogenic properties in hepatocellular carcinoma through interaction with ASPH .

How are INPP5F antibodies typically generated for research applications?

INPP5F antibodies can be generated using several methodological approaches:

• GST-fusion protein method: As detailed in the literature, GST-INPP5F fusion protein can be expressed in bacteria (typically BL21 strain), purified using standard techniques, and used to immunize rabbits . Specifically, a region encoding C-terminal amino acids is amplified by PCR, cloned into a vector such as pGEX-2T, and the fusion protein is purified using Glutathione Sepharose 4B .

• Commercial antibodies: Both monoclonal mouse anti-human INPP5F antibodies (e.g., Abcam ab236391, used at 1:200 dilution for IHC) and rabbit polyclonal antibodies have been validated in research contexts .

For antibody purification, techniques such as Melon Gel IgG Spin Purification can be employed prior to experimental use .

What expression patterns of INPP5F have been observed in normal and disease tissues?

INPP5F demonstrates distinct expression patterns that vary by tissue type and disease state:

Notably, INPP5F shows an intriguing subcellular localization pattern - being commonly nuclear-located in cells of adjacent non-tumor tissues, while predominantly cytoplasm-located in HCC .

What are the optimal protocols for Western blot detection of INPP5F?

For effective Western blot detection of INPP5F, researchers should consider these methodological details:

• Sample preparation: Tissues or cells should be lysed in buffer containing protease and phosphatase inhibitors (e.g., 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, with inhibitors including leupeptin, sodium pyrophosphate, Na₃VO₄, β-glycerophosphate, and PMSF) .

• Antibody selection and dilution:

  • For purified rabbit polyclonal antibodies, a 1:100 dilution in 5% milk is recommended

  • Commercial monoclonal antibodies may have different optimal dilutions (verify with manufacturer)

• Detection system: Visualization systems such as the Western Breeze Kit (Invitrogen) have been successfully employed in published research .

• Controls: Include appropriate positive and negative controls, particularly when studying INPP5F in new tissue types. Knockout tissues/cells provide excellent negative controls when available.

What are the recommended immunohistochemistry (IHC) protocols for INPP5F detection?

The following methodological considerations are important for IHC detection of INPP5F:

• Tissue preparation: Formalin-fixed, paraffin-embedded tissue sections are commonly used. After deparaffinization, H₂O₂ treatment and non-specific antigen blocking are essential steps .

• Antibody incubation: Monoclonal mouse anti-human INPP5F (1:200 dilution, e.g., Abcam ab236391) has been validated for IHC applications with overnight incubation at 4°C .

• Detection and visualization: After secondary antibody incubation, DAB staining is typically used for visualization .

• Scoring system: INPP5F expression can be scored based on staining intensity and percentage of positive cells. In published research, high expression has been defined as a total score greater than 4 points .

• Validation: Two independent pathologists should ideally assess staining to ensure scoring reliability .

How can researchers investigate INPP5F interactions with other proteins?

To study INPP5F protein interactions, several methodological approaches have proven effective:

• Immunoprecipitation assay: This technique can identify direct protein-protein interactions, as demonstrated in studies of INPP5F-ASPH interaction in HCC .

• Mass spectrometry analysis: Following immunoprecipitation, mass spectrometry can identify novel binding partners of INPP5F .

• Immunofluorescence co-localization: This approach visualizes spatial relationships between INPP5F and potential interaction partners within cells .

• Transcriptome-sequencing analysis: This method can identify genes whose expression is affected by INPP5F modulation, suggesting potential regulatory relationships .

When investigating specific pathways, researchers should consider examining known downstream targets, such as c-MYC, cyclin E1, and glycolysis-related genes in cancer studies, or Akt and GSK3β in cardiac research .

How can INPP5F antibodies be used to investigate cardiac hypertrophy mechanisms?

INPP5F antibodies are valuable tools for studying cardiac hypertrophy through several experimental approaches:

• Comparative expression analysis: Comparing INPP5F protein levels between normal hearts and hypertrophic models using Western blot analysis. In published research, INPP5F knockout mice showed augmented hypertrophy in response to stress compared to wild-type littermates .

• Signaling pathway investigation: INPP5F modulates the PI3K/Akt pathway in cardiac tissue. Researchers can use antibodies against INPP5F alongside phospho-Gsk3β (Ser 9), total Gsk3β, phospho-Akt (Ser 473), and total Akt to examine this signaling axis .

• Transgenic models: Both knockout and cardiac-specific overexpression models have been developed for INPP5F. Antibodies can validate the absence or overexpression of INPP5F in these models .

• Stress response studies: Using isoproterenol (ISO) treatment or other stress models, researchers can examine how INPP5F expression changes in response to hypertrophic stimuli .

• Histological correlation: IHC with INPP5F antibodies can be combined with wheat germ agglutinin (WGA) staining to correlate INPP5F expression with cellular hypertrophy measurements .

What are the experimental approaches for studying INPP5F's role in cancer progression?

To investigate INPP5F's functions in cancer, researchers can implement these methodological strategies:

• Expression profiling across cancer types: INPP5F shows divergent roles across cancer types. In HCC, it is frequently upregulated and associated with poor prognosis , while in gliomas, it appears to act as a tumor suppressor . Antibody-based techniques like IHC and Western blotting are essential for characterizing these expression patterns.

• Knockdown and overexpression studies:

  • For knockdown experiments, shRNA targeting INPP5F followed by Western blot validation has been effective

  • For overexpression, lentiviral vectors carrying INPP5F cDNA can be employed

• In vivo tumor models:

  • Subcutaneous xenograft models using cells with stable INPP5F modulation

  • Orthotopic tumor models (e.g., injecting cells under liver capsule for HCC studies)

• Functional assays:

  • Proliferation assays (CCK-8, EdU incorporation)

  • Cell cycle analysis (flow cytometry)

  • Colony formation assays

  • Metabolism assays (glucose consumption, lactate production)

• Downstream target identification: Using IHC with antibodies against potential targets (e.g., c-MYC, cyclin E1, Ki-67) in tumor tissues to correlate with INPP5F expression .

How can researchers investigate the nuclear versus cytoplasmic localization of INPP5F?

The differential subcellular localization of INPP5F between normal and cancer tissues presents an intriguing research area. Methodological approaches include:

• Subcellular fractionation: Separating nuclear and cytoplasmic fractions followed by Western blot analysis of INPP5F distribution.

• Immunofluorescence microscopy: Using INPP5F antibodies with nuclear counterstains (DAPI) to visualize localization patterns. This approach has revealed that INPP5F is commonly nuclear-located in cells of adjacent non-tumor tissues, while cytoplasm-located in HCC .

• Identification of nuclear localization/export signals: Computational and mutational analysis of INPP5F sequence to identify regulatory elements controlling its subcellular distribution.

• Stimulation experiments: Treating cells with various stimuli to determine if INPP5F translocation can be triggered experimentally.

• Co-localization studies: Combining INPP5F antibodies with markers for specific subcellular compartments to precisely map its distribution.

How can researchers address specificity concerns with INPP5F antibodies?

Ensuring antibody specificity is critical for reliable INPP5F research. Recommended validation approaches include:

• Knockout/knockdown controls: Testing antibodies on samples with confirmed INPP5F knockout or knockdown. Published research has validated antibodies using shRNA-mediated INPP5F knockdown .

• Overexpression controls: Testing on samples with confirmed INPP5F overexpression.

• Peptide competition assays: Pre-incubating antibody with purified INPP5F peptide to block specific binding.

• Multiple antibody verification: Using different antibodies targeting distinct INPP5F epitopes to confirm consistent results.

• Cross-reactivity assessment: Testing antibodies against related phosphatases to ensure specificity.

• Purification techniques: Using methods like Melon Gel IgG Spin Purification to minimize non-specific binding .

How should researchers interpret contradictory findings about INPP5F function in different disease models?

INPP5F shows context-dependent functions across different disease models, requiring careful interpretation:

• Tissue-specific effects: INPP5F appears to have different roles in different tissues - cardioprotective in heart tissue , oncogenic in HCC , prognostic in CLL , and tumor-suppressive in gliomas .

• Mechanistic differences: The molecular mechanisms differ by context:

  • In cardiac tissue: INPP5F modulates PI3K/Akt signaling to regulate hypertrophy

  • In HCC: INPP5F interacts with ASPH to activate Notch signaling and upregulate c-MYC and cyclin E1

  • In gliomas: INPP5F inhibits STAT3 signaling

• Analytical approach for reconciling contradictions:

  • Catalogue specific differences in experimental systems (cell types, disease models)

  • Consider expression levels in different contexts

  • Examine interaction partners unique to each tissue type

  • Investigate pathway differences and tissue-specific signaling networks

  • Consider subcellular localization (nuclear vs. cytoplasmic) as this may explain functional differences

• Integrated hypothesis development: When confronted with contradictory data, develop models that accommodate tissue-specific functions rather than seeking a universal mechanism.

What controls are essential when studying INPP5F in complex biological systems?

To ensure robust INPP5F research, the following controls are essential:

• Expression controls:

  • Positive control tissues with known INPP5F expression

  • Negative control tissues with minimal INPP5F expression

  • Isotype controls for antibody specificity

• Genetic controls:

  • INPP5F knockout or knockdown samples

  • INPP5F overexpression systems

• Pathway controls:

  • When studying INPP5F in PI3K/Akt pathway, include analysis of phospho-Akt and GSK3β

  • For cell cycle studies, include c-MYC and cyclin E1 analysis

• Interaction controls:

  • When studying protein interactions, include both co-immunoprecipitation and reverse co-immunoprecipitation

  • Use unrelated proteins as negative controls for interaction specificity

• Quantification controls:

  • For IHC scoring, use multiple independent observers

  • For cell size measurement (in hypertrophy studies), ensure adequate cell sampling (hundreds of cells from multiple hearts)

By implementing these methodological controls, researchers can generate more reliable and reproducible data when studying the complex biological functions of INPP5F.