FNIP1 Antibody, FITC conjugated

What is FNIP1 and what cellular functions does it regulate?

FNIP1 (Folliculin-interacting protein 1) is a cytoplasmic protein that functions as a binding partner of the GTPase-activating protein FLCN and plays crucial roles in multiple cellular pathways. It is primarily involved in the cellular response to amino acid availability by regulating the non-canonical mTORC1 signaling cascade that controls MiT/TFE transcription factors TFEB and TFE3 . FNIP1 is required to promote FLCN recruitment to lysosomes and interaction with Rag GTPases, leading to activation of non-canonical mTORC1 signaling . Additionally, FNIP1 and FLCN together regulate autophagy, with FNIP1 interacting with GABARAP following phosphorylation by ULK1 . FNIP1 also functions as a co-chaperone of HSP90AA1/Hsp90 and is involved in energy and nutrient sensing through the AMPK and mTOR signaling pathways .

What are the optimal storage conditions for FNIP1 Antibody, FITC conjugated to maintain its activity?

The FNIP1 Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt to maintain optimal activity . The antibody is shipped at 4°C, but long-term storage requires lower temperatures . It is critical to avoid repeated freeze-thaw cycles as this can significantly compromise antibody performance . The antibody is supplied in a storage buffer containing 0.03% Proclin 300 as a preservative, along with 50% glycerol and 0.01M PBS at pH 7.4, which helps maintain stability during storage . When working with the antibody, it is advisable to aliquot it upon receipt to minimize the number of freeze-thaw cycles each portion undergoes.

What is the specificity and cross-reactivity profile of commercially available FNIP1 antibodies?

The FITC-conjugated FNIP1 antibody is specifically raised against recombinant human Folliculin-interacting protein 1 protein (amino acids 221-508) and demonstrates reactivity with human FNIP1 . While some FNIP1 antibodies show cross-reactivity with mouse and rat samples, the FITC-conjugated variant has been primarily tested and confirmed for human reactivity . Other non-conjugated FNIP1 antibodies exhibit broader cross-reactivity, with some variants reacting with mouse, human, and rat samples . Strong expression of FNIP1 is typically found in heart, liver, placenta, muscle, nasal mucosa, salivary gland, and uvula tissues, with moderate expression in kidney and lung . Notably, higher FNIP1 levels have been detected in clear cell renal cell carcinoma and chromophobe RCC compared to normal kidney tissue .

How should researchers design control experiments when studying FNIP1 in relation to mTORC1 signaling and autophagy?

When studying FNIP1's role in mTORC1 signaling and autophagy, researchers should implement multiple control strategies. First, include nutrient-replete and nutrient-depleted conditions since FNIP1 functions differently based on amino acid availability . In low-amino acid conditions, FNIP1 becomes part of the lysosomal folliculin complex (LFC) that inhibits FLCN's GTPase-activating activity, whereas amino acid restimulation causes LFC complex disassembly .

Second, incorporate genetic controls including FNIP1 knockout cells alongside wild-type cells, as Fnip1-deficient B cell progenitors show increased nuclear localization of TFE3, increased expression of TFE3-target genes, increased lysosome numbers and function, and enhanced autophagic flux . To specifically assess mTORC1 involvement, include conditions with rapamycin treatment to inhibit mTORC1 signaling .

Third, design experiments to monitor lysosomal localization of FNIP1 and FLCN under various conditions, as inappropriate mTOR localization at the lysosome under nutrient-depleted conditions has been observed in FNIP1-deficient cells . Finally, include markers for both AMPK activation and mTORC1 activation, as both pathways are activated in FNIP1-deficient cells .

What experimental approaches can distinguish between FNIP1-dependent and FNIP1-independent mechanisms in cellular stress responses?

To distinguish between FNIP1-dependent and FNIP1-independent mechanisms in cellular stress responses, researchers should employ a multi-faceted experimental strategy. Begin with genetic manipulation approaches, creating FNIP1 knockdown/knockout models alongside rescue experiments using wild-type and mutant FNIP1 constructs . This allows for identification of phenotypes specifically attributable to FNIP1 loss.

Employ nutrient and energy stress paradigms that specifically activate AMPK or inhibit mTORC1, as FNIP1 is known to integrate signals from both pathways . Ex vivo lysine or arginine depletion experiments are particularly informative, as FNIP1-deficient cells show increased apoptosis under these conditions .

Utilize pharmacological inhibitors targeting specific nodes in FNIP1-regulated pathways (AMPK inhibitors, mTORC1 inhibitors) to determine pathway dependencies . Important findings from previous studies showed that genetic inhibition of AMPK or inhibition of mTORC1 failed to rescue B cell development in FNIP1-deficient mice, indicating additional FNIP1 functions beyond these pathways .

Incorporate transcriptional profiling to identify FNIP1-dependent gene expression changes, particularly focusing on TFE3-target genes which are upregulated in FNIP1-deficient cells . Finally, employ phosphoproteomic analyses to track signaling changes, focusing on AMPK and mTORC1 substrates, as FNIP1 may regulate phosphorylation of proteins like RPS6KB1 .

What are the optimal conditions for using FNIP1 Antibody, FITC conjugated in immunofluorescence studies of lysosomal positioning and mTORC1 signaling?

When using FNIP1 Antibody, FITC conjugated for immunofluorescence studies focusing on lysosomal positioning and mTORC1 signaling, several technical parameters require optimization. The antibody has been Protein G purified to >95% purity, providing high specificity for the target . For co-localization studies examining FNIP1 interaction with lysosomal components or mTORC1 machinery, the following protocol is recommended:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.1% Triton X-100 for 10 minutes.

• Block with 5% normal serum in PBS for 1 hour to reduce background signals.

• Dilute the FNIP1 Antibody, FITC conjugated at 1:100 to 1:500 in blocking buffer (optimal dilution should be determined empirically for each experimental system).

• For co-localization studies, combine with antibodies against lysosomal markers (LAMP1, LAMP2) or mTORC1 components (mTOR, Raptor) labeled with spectrally distinct fluorophores.

• For studying nutrient-dependent dynamics, prepare parallel samples with complete media or media lacking specific amino acids for 1-4 hours before fixation, as FNIP1 localization changes with amino acid availability .

• Include controls for antibody specificity using FNIP1 knockdown cells.

• Employ confocal microscopy with appropriate filter settings for FITC (excitation ~495 nm, emission ~520 nm) to minimize bleed-through when performing co-localization studies.

• For quantitative analysis, measure co-localization coefficients between FNIP1 and markers of interest under different experimental conditions.

This approach will enable visualization of FNIP1's dynamic localization in response to nutrient availability and its spatial relationship with the mTORC1 signaling machinery.

What modifications to standard ELISA protocols are necessary when using FNIP1 Antibody, FITC conjugated for quantitative analysis?

When adapting standard ELISA protocols for quantitative analysis using FNIP1 Antibody, FITC conjugated, several important modifications are necessary to optimize performance and ensure reliable results:

• Detection System Adjustment: Unlike standard ELISA protocols that typically use HRP-conjugated secondary antibodies, the FITC conjugation enables direct fluorescence detection. Use fluorescence plate readers with appropriate excitation (~495 nm) and emission (~520 nm) filters for FITC detection .

• Plate Selection: Use black or white opaque microplates instead of transparent plates to enhance fluorescence signal detection and minimize background interference.

• Standard Curve Generation: Develop a standard curve using recombinant FNIP1 protein (ideally matching the immunogen region aa 221-508) to enable accurate quantification . Plot fluorescence intensity against known concentrations of recombinant FNIP1.

• Signal Amplification Considerations: Unlike enzyme-based detection systems that provide signal amplification, FITC provides direct fluorescence without amplification. Therefore, more primary antibody may be required (typically 2-5 μg/mL for coating) compared to traditional ELISA protocols.

• Photobleaching Precautions: Protect the assay from light exposure during all incubation steps to prevent FITC photobleaching, which can reduce signal intensity.

• Buffer Optimization: The antibody is provided in a buffer containing 50% glycerol and 0.01M PBS at pH 7.4 . For ELISA, dilute in carbonate-bicarbonate buffer (pH 9.6) for coating steps and in PBS with 1% BSA for subsequent steps.

• Blocking Considerations: Use BSA-based blocking buffers rather than milk-based ones, as milk can sometimes contribute to higher background with fluorescence-based detection.

• Quality Control Measures: Include controls for autofluorescence from sample matrix and incorporate a calibration step to account for day-to-day variations in instrument performance.

How should researchers interpret contradictory findings when studying FNIP1 involvement in AMPK and mTORC1 signaling pathways?

When faced with contradictory findings regarding FNIP1's involvement in AMPK and mTORC1 signaling, researchers should consider several factors that might explain the discrepancies. First, examine the cellular context carefully, as FNIP1 functions differently across cell types. For instance, its role in B cell development appears to be independent of AMPK and mTORC1, despite both pathways being activated in FNIP1-deficient B cell progenitors .

Second, consider the nutrient status and experimental conditions. FNIP1's function changes dramatically between amino acid-replete and amino acid-depleted states . In low-amino acid conditions, FNIP1 is part of the lysosomal folliculin complex that inhibits FLCN's GTPase-activating activity, while amino acid restimulation leads to complex disassembly and activation of mTORC1 .

Third, evaluate the involvement of additional regulators. FNIP1 interacts with multiple proteins beyond AMPK and mTORC1 components, including HSP90AA1/Hsp90, where it acts as a co-chaperone . These interactions may dominate in certain contexts, explaining why manipulating AMPK or mTORC1 alone fails to rescue FNIP1-deficient phenotypes .

Fourth, consider post-translational modifications. FNIP1 undergoes phosphorylation by multiple kinases including ULK1 and CK2, which affects its function . Differences in these modifications across experimental systems could lead to seemingly contradictory results.

Finally, researchers should analyze whether the observed contradictions stem from genuine biological complexity or technical limitations such as antibody specificity, the sensitivity of detection methods, or timing of analyses within dynamic signaling cascades.

What quantitative approaches are most appropriate for analyzing FNIP1's role in autophagy and lysosomal function?

To quantitatively analyze FNIP1's role in autophagy and lysosomal function, researchers should implement a comprehensive multi-parameter approach. Based on findings that FNIP1-deficient B cell progenitors exhibit increased nuclear localization of TFE3, enhanced expression of TFE3-target genes, increased lysosome numbers and function, and increased autophagic flux , the following analytical methods are recommended:

Table 1: Quantitative Approaches for Analyzing FNIP1's Role in Autophagy and Lysosomal Function

For time-course experiments, apply principal component analysis or hierarchical clustering to identify temporal patterns in the data. Compare FNIP1 wild-type versus knockout conditions using appropriate statistical tests, accounting for biological replicates. Incorporate nutrient availability as an experimental variable, as FNIP1's function changes with amino acid levels .

When analyzing protein-protein interactions between FNIP1 and autophagy machinery components like GABARAP , quantify interaction strength using co-immunoprecipitation followed by densitometry or more advanced techniques like proximity ligation assays with statistical analysis of puncta formation.

What are the emerging research directions for studying FNIP1's role in B cell development independent of p53 and Bcl-xL pathways?

Recent findings have established that FNIP1 is required for pre-B cell development and survival independent of p53 and Bcl-xL pathways , opening several promising research directions. Investigators should explore the metabolic checkpoint hypothesis, as FNIP1-deficient B cell progenitors show dysregulated energy metabolism with both AMPK and mTORC1 simultaneously activated . This unusual metabolic state may represent a novel checkpoint in B cell development.

The role of TFE3 nuclear localization in B cell development warrants in-depth investigation, as FNIP1-deficient B cell progenitors exhibit increased nuclear TFE3 and enhanced expression of TFE3-target genes . Researchers should map the comprehensive TFE3-dependent transcriptional program in developing B cells and determine which targets are critical for B cell survival.

Amino acid sensing appears to be a key function of FNIP1, as demonstrated by increased apoptosis of FNIP1-deficient cells during ex vivo lysine or arginine depletion . Future work should characterize the amino acid-sensing mechanisms in B cell progenitors and determine how these integrate with developmental signals specific to B lymphopoiesis.

The increased autophagy and lysosomal function observed in FNIP1-deficient cells suggests another research direction focused on organelle quality control during B cell development . Studies should address whether precise regulation of autophagy timing and magnitude is essential for proper B cell maturation.

Finally, given that genetic inhibition of AMPK, inhibition of mTORC1, or restoration of cell viability with a Bcl-xL transgene all failed to rescue B cell development in FNIP1-deficient mice , researchers should perform unbiased screens to identify the critical pathways through which FNIP1 supports B cell development.

How can researchers leverage FITC-conjugated FNIP1 antibodies to study dynamic changes in protein localization during nutrient stress responses?

FITC-conjugated FNIP1 antibodies offer unique advantages for investigating dynamic changes in FNIP1 localization during nutrient stress. For live-cell imaging studies, researchers can employ membrane-permeabilizing techniques like digitonin permeabilization followed by introduction of FITC-conjugated FNIP1 antibodies to track protein relocalization in real-time during nutrient shifts . This approach allows visualization of the dynamic movement of FNIP1 between cytoplasmic pools and lysosomal membranes as amino acid availability changes.

For analysis of rapid relocalization events, researchers can develop a pulse-chase immunofluorescence protocol using the FITC-conjugated antibody . This involves fixing cells at short time intervals following nutrient depletion or restoration, allowing construction of a high-resolution temporal map of FNIP1 movement within the cell.

To study co-trafficking with partner proteins, multi-color imaging can be performed combining FITC-conjugated FNIP1 antibody with differently labeled antibodies against FLCN, Rag GTPases, or components of the mTORC1 machinery . This approach reveals the sequence of protein recruitment to lysosomes and other subcellular compartments during nutrient stress.

Super-resolution microscopy techniques such as STORM or PALM can be applied using the FITC-conjugated FNIP1 antibody to achieve nanometer-scale resolution of FNIP1 localization relative to lysosomal subdomains . This approach can reveal previously undetectable organizational patterns on lysosomal membranes.

Correlative light and electron microscopy (CLEM) can be employed by using the FITC signal from labeled FNIP1 to identify regions of interest for subsequent electron microscopy analysis, providing ultrastructural context for FNIP1 localization during nutrient stress.

Finally, researchers can develop FRET-based assays combining the FITC-conjugated FNIP1 antibody with appropriate acceptor fluorophore-labeled antibodies against interaction partners to measure not just co-localization but direct protein-protein interactions as they change during nutrient stress responses .