APT1 Antibody

What is APT1 and why is it important in cellular biology?

APT1 (also known as LYPLA1 or LPL-I) functions as both an acyl-protein thioesterase and a lysophospholipase. It hydrolyzes fatty acids from S-acylated cysteine residues in proteins such as trimeric G alpha proteins and HRAS. APT1 also catalyzes depalmitoylation of proteins including ADRB2, KCNMA1, and SQSTM1 . As a negative regulator of autophagy, it mediates palmitoylation of SQSTM1, which decreases affinity between SQSTM1 and ATG8 proteins . In addition to its thioesterase activity, APT1 hydrolyzes lysophosphatidylcholine (lyso-PC) and other lysophospholipids, though its thioesterase activity is significantly higher than its lysophospholipase activity .

What are the validated applications for APT1 antibodies?

Based on current research, APT1 antibodies have been validated primarily for Western blotting (WB) applications with demonstrated reactivity against human, mouse, and rat samples . Notably, the rabbit recombinant monoclonal antibody against Lysophospholipase 1/LPL-I (clone EPR3667) has been cited in at least 15 publications . When selecting an APT1 antibody, confirm that it has been validated for your specific species of interest and application through product documentation or published literature.

How can I verify APT1 antibody specificity?

The gold standard for antibody specificity validation is comparative analysis using wild-type and knockout samples. For example, search result #7 demonstrates a validation approach where the antibody (ab91606) was tested against wild-type HEK-293T cell lysate alongside LYPLA1 knockout HEK-293T cell lysate . The antibody detected a band at the predicted size (25 kDa) only in wild-type samples and not in knockout samples, confirming its specificity. For proper controls, include appropriate loading controls (such as GAPDH) and ensure reducing conditions when indicated in the antibody documentation.

What is the recommended protocol for using APT1 antibodies in Western blotting?

Based on published protocols, a typical Western blotting procedure for APT1 detection includes:

• Sample preparation in RIPA lysis buffer with 1 mM phenylmethylsulfonyl fluoride

• Loading equal amounts of protein onto 8-16% Bis-Tris gels

• Transferring separated proteins to PVDF membranes

• Blocking with appropriate buffer

• Incubating with anti-APT1 primary antibody at manufacturer-recommended dilutions (e.g., 1/5000 for ab91606)

• Washing and incubating with secondary antibody (e.g., Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity)

• Visualizing protein signals using enhanced chemiluminescence (ECL) detection reagents

• Quantifying bands using image analysis software like ImageJ

For optimal results, always verify antibody-specific recommendations for incubation times, buffer compositions, and blocking reagents.

How can I detect phosphorylated APT1 in cell lysates?

Detecting phosphorylated APT1 requires phospho-specific antibodies. According to search result #6, researchers have successfully generated and validated phospho-specific antibodies to both serine 209 and serine 210 of APT1 . The methodology involves:

• Treating cells with stimuli that induce APT1 phosphorylation (e.g., Wnt5a for 15 minutes)

• Immunoprecipitating APT1 (if needed for enrichment)

• Separating proteins by SDS-PAGE

• Immunoblotting with phospho-APT1 antibodies (pS209-APT1 or pS210-APT1)

• Comparing phosphorylation levels between treatment conditions

For high stoichiometry phosphorylation events (like pS210-APT1 after Wnt5a stimulation), detection may be possible directly in whole cell lysate without immunoprecipitation .

What methods can be used to measure APT1 enzymatic activity?

The depalmitoylation probe DPP-3 provides a powerful tool for measuring APT1 activity both in vitro and in live cells. This fluorescent probe contains a thiol-conjugated seven-carbon fatty acid that, when hydrolyzed, generates a fluorescent product measurable at λex490/9 nm; λem545/20 nm .

In vitro protocol:

• Express and purify 6x His-tagged APT1 from E. coli

• Incubate purified APT1 with DPP-3 at various concentrations

• Measure relative fluorescence over time

• Calculate initial velocities at multiple substrate concentrations

• Compare activity between wild-type and mutant APT1 variants

Live-cell protocol:

• Transfect cells with APT1 constructs of interest

• Incubate cells with DPP-3 probe

• Measure fluorescence emission by live-cell microscopy at defined timepoints

• Quantify fluorescence in individual cells

• Compare activity between conditions or APT1 variants

How does phosphorylation affect APT1 activity and how can this be studied?

APT1 phosphorylation at serine residues 209 and 210 significantly increases its depalmitoylating activity. Studies have mapped these regulatory phosphorylation sites using mass spectrometry after immunoprecipitation . The impact of phosphorylation can be investigated through several approaches:

• Site-directed mutagenesis: Generate phosphomimetic (S209D) and phospho-deficient (S209A) mutants

• Activity assays: Compare depalmitoylating activity between wild-type and mutant APT1 using the DPP-3 probe

• Kinase inhibition: Treat cells with kinase inhibitors (e.g., BI-D1870 or staurosporine) to block phosphorylation

• Stimulation experiments: Activate signaling pathways (e.g., Wnt5a) that induce APT1 phosphorylation

Research has shown that phosphomimetic APT1 S209D exhibits higher initial velocities at all substrate concentrations compared to wild-type APT1 , as demonstrated in the following table:

What are the considerations for using APT1 antibodies in co-immunoprecipitation studies?

When designing co-immunoprecipitation (co-IP) experiments to study APT1 interactions:

• Antibody selection: Choose antibodies specifically validated for immunoprecipitation

• Expression system: Consider using tagged APT1 (e.g., CFP-FLAG tagged) to facilitate clean immunoprecipitation

• Lysis conditions: Optimize lysis buffers to preserve protein-protein interactions while effectively solubilizing membrane-associated complexes

• Elution method: Use specific elution with FLAG peptide for tagged constructs to reduce non-specific binding

• Controls: Include appropriate negative controls (non-specific IgG, knockout cells)

• Validation: Confirm pull-down efficiency by Western blotting for APT1

• Mass spectrometry analysis: For identifying novel interaction partners, consider analysis of immunoprecipitated complexes by LC-MS/MS

Research has identified over 200 shared interactors between APT1 and APT2, many of which localize to membranes and contain pleckstrin homology-like domains involved in lipid binding .

What approaches can resolve contradictory findings about APT1 substrate specificity?

Contradictions in reported APT1 substrate specificity might stem from differences in experimental conditions, cell types, or assay systems. To resolve such discrepancies:

• Comparative substrate profiling: Perform palmitoyl-proteomics assays across multiple systems

• Genetic models: Utilize APT1 knockout models (e.g., APT1LKO mice) to identify accumulated palmitoylated proteins in vivo

• Proximity labeling: Employ APT1-mTurquoise BioID fusion proteins to identify proteins in proximity to APT1

• Stringency analysis: Apply different stringency criteria when analyzing mass spectrometry data (e.g., low-stringency vs. medium-stringency analysis as demonstrated in search result #4)

• Validation: Confirm mass spectrometry findings with orthogonal biochemical approaches

Research has shown that in liver tissue, APT1 deficiency is associated with increased palmitoylation of 128 proteins under low-stringency analysis criteria, compared to only 4 proteins for APT2 deficiency, suggesting preferential depalmitoylation by APT1 in this tissue .

What is the role of APT1 in atherosclerosis development?

Recent research has identified APT1 as a crucial factor in atherosclerosis development. In an in vitro atherosclerosis cell model using oxidative low-density lipoprotein (ox-LDL), APT1 expression levels were significantly increased compared to normal controls . Additionally, APT1 localization shifted to the plasma membrane in atherosclerotic conditions .

In vivo studies using ApoE-/- mice fed a Western diet showed increased APT1 expression in multiple organs including the aorta, heart, and liver compared to control groups . This suggests endothelial-derived APT1-mediated macrophage-endothelial cell interactions may participate in atherosclerosis development by regulating the Ras/MAPK signaling pathway .

Researchers investigating this pathway should consider:

• Expression analysis in both in vitro and in vivo models

• Subcellular localization studies

• Pathway analysis focusing on Ras/MAPK signaling

• Therapeutic targeting potential

How is APT1 involved in biopharmaceutical product stability?

A groundbreaking study has identified APT1 as a polysorbate-degrading host cell protein (HCP) in monoclonal antibody (mAb) formulations . Polysorbate is critical for maintaining protein stability during a drug product's shelf life but is vulnerable to breakdown by residual HCPs with hydrolytic activity .

Using activity-based protein profiling (ABPP) coupled with mass spectrometry, researchers identified APT1 as the specific HCP responsible for polysorbate degradation in a mAb formulation experiencing stability issues . The role of APT1 was validated by:

• Matching the polysorbate degradation fingerprint in the mAb formulation with that of recombinant APT1

• Finding correlation between APT1 levels and polysorbate degradation rates

• Successfully halting polysorbate degradation using APT1-specific inhibitors ML348 and ML211

This research provides important considerations for biopharmaceutical development and quality control, particularly for antibody-based therapeutics where APT1 contamination could impact product stability.

How can the interaction between APT1 and the Wnt signaling pathway be experimentally investigated?

The discovery that Wnt5a signaling induces APT1 phosphorylation, increasing its depalmitoylating activity, opens new research directions. To investigate this interaction:

• Phosphorylation site mapping: Use mass spectrometry to identify and quantify phosphorylation at serine residues 209 and 210 after Wnt5a stimulation

• Activity assays: Measure APT1 activity using the DPP-3 probe before and after Wnt5a treatment

• Signaling inhibition: Block Wnt5a-induced APT1 activation using kinase inhibitors

• Mutational analysis: Compare wild-type APT1 response to Wnt5a with phospho-mutants

• Functional outcomes: Assess biological consequences such as melanoma invasion capacity

Research has shown that Wnt5a treatment increases APT1 WT activity even beyond levels observed with the phosphomimetic mutant APT1 S209D, suggesting additional regulation mechanisms . This pathway has implications for cancer biology, as APT1 phosphorylation correlates with increased tumor grade and metastasis in melanoma .

What are the current methods for detecting APT1 in ELISA/ELONA systems?

While standard ELISA systems typically use antibodies as both capture and detection agents, recent research has explored aptamer-based approaches (ELONA - Enzyme-Linked Oligonucleotide Assay) that may have applications for APT1 detection. Key methodological considerations include:

• Capture agent concentration: Signal intensities increase with higher concentrations of capture agent (from 2.5 to 20 nM)

• Aptamer format: Bivalent aptamers (V-Apt) exhibit higher detection sensitivities and signal intensities than univalent aptamers (I-Apt)

• Sample matrix effects: High-affinity aptamers remain robust under serum conditions (10%), while low-affinity aptamers show reduced signal intensities

• Optimization for blood tests: Special consideration is needed when detecting antigens in human fluids, as serum or plasma may contain binders to the antigen that competitively inhibit detection