LARP4 Antibody

What is LARP4 and why is it significant in research?

LARP4 is an RNA-binding protein that interacts with poly(A) RNA both directly through its N-terminal region and indirectly via poly(A) binding proteins (PABPs) . It functions in several key cellular processes:

• Maintains poly(A) tail length by competing with deadenylases for PABP binding sites

• Associates with the 40S ribosomal subunit and polysomes to regulate mRNA translation

• Binds nuclear-encoded mitochondrial mRNAs (NEMmRNAs) to promote mitochondrial function

• Interacts with RACK1 at ribosomal sites through its conserved region-2 (CR2)

• Regulates cytoskeletal organization and cell migration

LARP4's diverse functions make it particularly relevant for cancer research, RNA metabolism studies, and mitochondrial biology research.

What experimental applications are LARP4 antibodies validated for?

LARP4 antibodies have been validated for multiple applications across diverse experimental systems:

When selecting antibodies, consider species reactivity (human, mouse, rat are most common) and the specific epitope location (e.g., C-terminal antibodies targeting aa 650-724) .

How should researchers validate LARP4 antibody specificity?

Validation should follow a multi-step approach:

• Knockout/knockdown controls: Generate LARP4 knockout cell lines using CRISPR-Cas9 with specific gRNAs (e.g., 5′-TAGACCGAGTACTGTTGGTG-3′ and 5′-TTGCGGCGGCGGGAACGATT-3′) . Compare antibody signals between wild-type and knockout samples.

• Overexpression verification: Express tagged versions (Flag-tagged or GFP-tagged LARP4) and confirm co-detection with anti-tag and anti-LARP4 antibodies .

• Size verification: Confirm detection at expected molecular weight (80.6 kDa for canonical isoform) .

• Cross-reactivity assessment: Test against multiple species if working with non-human models. LARP4 orthologs exist in mouse, rat, bovine, frog, chimpanzee and chicken species .

• Isoform consideration: Account for up to 7 different isoforms that have been reported for this protein .

What protocols are recommended for LARP4 immunoprecipitation experiments?

For optimal LARP4 immunoprecipitation, follow these methodological guidelines:

For FLAG-tagged LARP4 IP:

• Prepare cell lysate (500 μg total protein) in wash buffer (50 mM Tris pH 8.0, 75 mM NaCl, 0.05% NP-40)

• Wash 50 μl anti-Flag M2 magnetic bead slurry three times with 250 μl wash buffer

• Add lysate to beads in 340 μl total volume and incubate at 4°C for 2 hours

• Perform 5 washes with 250 μl wash buffer

• Resuspend beads in SDS-PAGE sample buffer (without β-mercaptoethanol)

• Heat samples at 95°C for 5 minutes, collect supernatant

• Add 5% fresh β-mercaptoethanol before loading

For endogenous LARP4 IP:

• Pre-incubate 20 μl Protein-A Sepharose beads with 10 μl anti-RACK1 antibody

• Add beads to 500 μg total cell protein extract

• Follow washing and elution steps as above

This approach has successfully demonstrated LARP4 interactions with RACK1 and PABP in published studies.

How can researchers effectively study LARP4's role in mRNA stability?

LARP4 significantly affects mRNA stability through poly(A) tail protection. To study this:

• Reporter assay system:

  • Co-transfect cells with FLAG-LARP4 and reporter constructs (e.g., β-globin or GFP mRNAs)

  • Include controls with and without AU-rich elements (AREs)

  • The HEK293 cell system with FLAG-LARP4-WT shows optimal expression at 3-4 fold higher than endogenous levels

• Decay measurement:

  • Use transcription inhibitors (e.g., actinomycin D) and collect samples at time intervals

  • Conduct northern blot or RT-qPCR analysis

  • Plot data with t=0 values set to 100%

• Results interpretation:
  Published data shows that LARP4 substantially increases mRNA stability:

  • β-globin mRNA shows ~50% decrease after 70 min with empty vector

  • With LARP4 WT, 50% decrease occurs at 240 min

  • LARP4 CS (codon-substituted) variant extends stability even further (~65% remaining at 240 min)

This methodology allows for quantification of LARP4's impact on different mRNA substrates.

How can researchers investigate LARP4's interaction with RACK1 and the ribosome?

LARP4 interacts with RACK1 through its conserved region-2 (CR2). To study this interaction:

• Mutation analysis approach:

  • Generate targeted mutations in the CR2 region (positions 615-625 in LARP4)

  • Create substitution mutants (designated R1) or deletion mutants (designated ΔRIR)

  • For LARP4B, target corresponding positions 646-656

• Validation methods:
  a. Yeast two-hybrid screening
  b. Co-immunoprecipitation assays:

  • Use anti-RACK1 antibody to pull down LARP4

  • Perform reciprocal IP with anti-FLAG to detect RACK1

  • Compare WT vs. mutant LARP4 association
    c. In vitro binding assays with purified recombinant proteins:

  • Generate pLIB-LARP4 constructs with C-terminal StrepII tag

  • Express and purify using standard methods

  • Use isothermal titration calorimetry (ITC) to determine binding constants

• Structural prediction:

  • AlphaFold2-Multimer predicts high-confidence interaction of CR2 with RACK1 propellers 5 and 6

Published results confirm that CR2 mutations strongly decrease LARP4 association with cellular RACK1 and ribosomes, while PABP association is less affected, consistent with independent interactions .

What approaches are effective for studying LARP4's role in mitochondrial function?

LARP4 binds and regulates nuclear-encoded mitochondrial mRNAs (NEMmRNAs). Study this function using:

• Target identification:

  • Perform enhanced crosslinking and immunoprecipitation (eCLIP)

  • Analyze RNA targets by next-generation sequencing

  • Published data shows 713 human mRNA targets for LARP4, with 186 (26%) encoding mitochondrial proteins

• Mitochondrial enrichment analysis:

  • Use APEX-seq atlas data to determine mitochondrial localization of LARP4 target mRNAs

  • LARP4 targets show higher mitochondrial enrichment compared to non-targets across all conditions

• Functional validation:
  a. Protein expression analysis:

  • Generate CRISPR-mediated LARP4^(-/-) knockout cells

  • Analyze protein expression of respiratory chain complex proteins (RCCPs) and mitochondrial ribosome proteins (MRPs)

  • HEK293 LARP4^(-/-) cells show significant reduction in multiple RCCPs:

    • NDUFA8: KO/WT = 0.67

    • NDUFB9: KO/WT = 0.74

    • COX6B1: KO/WT = 0.16

  • And MRPs:

    • MRPS5: KO/WT = 0.52

    • MRPL24: KO/WT = 0.72

  b. Mitochondrial function assays:

  • Measure oxidative phosphorylation capacity

  • Perform rescue experiments with LARP4 re-expression

These approaches have demonstrated that LARP4 binds and positively regulates NEMmRNAs to promote mitochondrial respiratory function.

How can LARP4 antibodies be utilized to investigate cell migration mechanisms?

LARP4 regulates cytoskeletal organization and cell migration. To study this role:

• Knockdown validation approach:

  • Perform siRNA-mediated knockdown of LARP4

  • Confirm knockdown efficiency by western blot using anti-LARP4 antibody (e.g., Proteintech 16529-1-AP)

• Migration assays:
  a. Modified wound healing assay:

  • Create cell-free gap using removable stopper

  • Track migration into the gap

  • LARP4-depleted PC3 cells show significantly higher migration

  b. Random motility assay:

  • Perform time-lapse microscopy

  • Track individual cell paths

  • Measure migration speed

  • LARP4 depletion results in significant increase in cell migration speed

• Interaction with cytoskeletal components:

  • Investigate LARP4's interaction with Filamin A (FLNA) using:

    • Co-immunoprecipitation with LARP4 antibodies

    • Proximity ligation assay (PLA)

    • Generate non-FLNA-binding mutant LARP4 (F277A)

    • Fluorescence recovery after photobleaching (FRAP) analysis of GFP-LARP4

These methodologies have revealed that LARP4 knockdown increases cell migration speed and expression of the F277A mutant LARP4 in LARP4-KD cells leads to higher cell migration speed compared to wild-type LARP4 .

How can researchers address inconsistent results between LARP4 antibodies in different applications?

Inconsistent results can stem from several factors:

• Epitope accessibility issues:

  • Different antibodies target distinct regions of LARP4 (N-terminal, La module, CR2, C-terminal)

  • Protein-protein interactions may mask epitopes in specific applications

  • Solution: Use multiple antibodies targeting different epitopes for validation

• Isoform specificity:

  • LARP4 has up to 7 different reported isoforms

  • Solution: Verify which isoform(s) your antibody detects using recombinant protein controls

• Cross-reactivity with LARP4B:

  • LARP4 and its paralog LARP4B share conserved regions

  • Solution: Validate specificity using knockout controls for both proteins

• Cell-type variation:

  • LARP4 expression varies across tissue types

  • Detection sensitivity may differ in HEK293 vs. U2OS cells

  • The level of target protein disruption observed by immunoblot is often less pronounced in U2OS LARP4 KO lines compared to HEK293

• Application-specific considerations:

  • For WB: Optimize lysis conditions (50 mM Tris pH 8.0, 75 mM NaCl, 0.05% NP-40 works well)

  • For IP: Pre-clearing lysates and longer incubation times (2 hours at 4°C) improve specificity

  • For IF: Fixation method affects epitope preservation (PFA vs. methanol)

What considerations are important when designing LARP4 functional rescue experiments?

Rescue experiments are critical for confirming phenotype specificity. Key considerations include:

• Expression level control:

  • LARP4 overexpression at 3-4 fold above endogenous levels extends mean transcriptome-wide poly(A) tails by 4 nt

  • LARP4 genetic deletion shortens mean poly(A) tail length by 5 nt

  • Use inducible systems to achieve physiological expression levels

• Mutant design strategy:

  • Target specific functional domains:

    • PAM2w (PABP interaction)

    • CR2 (positions 615-625, RACK1 interaction)

    • FLNA interaction (F277A mutation)

  • Use codon substitution (CS) variants to dissect non-protein functions

• Control constructs:

  • Include domain deletion controls (e.g., ΔRIR)

  • Use partial substitution mutants (e.g., L4B-R1 Par with R646E, K647E mutations)

• Readout selection:

  • mRNA stability: β-globin or GFP reporters with/without AREs

  • Translation efficiency: Luciferase reporters

  • Cell migration: Wound healing or random motility assays

  • Mitochondrial function: Oxidative phosphorylation capacity

This approach allows for robust attribution of phenotypes to specific LARP4 functions and domains.

How can researchers explore LARP4's potential roles in disease processes?

LARP4's involvement in fundamental cellular processes suggests potential roles in disease:

• Cancer research approaches:

  • Compare LARP4 expression in tumor vs. normal tissues using validated antibodies

  • Analyze LARP4 expression in metastatic vs. non-metastatic cell lines

  • Perform migration/invasion assays with LARP4 modulation

  • LARP4 depletion increases migration in PC3 and MDA-MB-231 cancer cell lines

• Mitochondrial disease investigations:

  • Compare LARP4 binding to NEMmRNAs in patient vs. control samples

  • Analyze if LARP4 dysfunction affects mitochondrial protein expression

  • Target verification should focus on respiratory chain complex proteins (RCCPs) and mitochondrial ribosome proteins (MRPs)

• Translation dysregulation studies:

  • Examine LARP4-ribosome interactions through RACK1 in stress conditions

  • Investigate how LARP4 mutations affect mRNA deadenylation in disease models

  • CR2 mutations decrease LARP4's ability to stabilize mRNAs containing AU-rich elements

These research directions leverage validated antibodies to explore LARP4's contributions to pathological processes.

What cutting-edge methods can advance understanding of LARP4 RNA-binding specificity?

To elucidate the RNA-binding specificity of LARP4:

• High-throughput binding assays:

  • RNA-compete to identify preferred binding motifs

  • CLIP-seq variations to map in vivo binding sites

  • LARP4 eCLIP data shows coating of many target mRNAs from CDS to 3′ UTR with highest peak density centered around the stop codon

• Structural biology approaches:

  • Cryo-EM of LARP4-ribosome complexes to visualize RACK1 interaction

  • NMR studies of the La module and N-terminal RNA-binding domains

  • X-ray crystallography of LARP4 bound to RNA substrates

• Proximity-dependent methods:

  • APEX-seq for spatial transcriptomics of LARP4-associated RNAs

  • The APEX-seq atlas shows higher mitochondrial enrichment for LARP4 targets compared to non-targets

  • BioID for identifying proximal proteins in different subcellular locations

• Machine learning applications:

  • Train models to predict LARP4 binding sites based on eCLIP data

  • Develop algorithms to predict functional consequences of LARP4 binding

These methodologies will help resolve how LARP4 achieves RNA-target specificity through its RNA-binding domains and protein interaction domains.