larp7 Antibody

What is LARP7 and what are its primary biological functions?

LARP7 is an RNA-binding protein characterized by a La module (consisting of a La-motif and RNA recognition motif) that enables specific RNA interactions. Its primary functions include:

• Acting as a core component of the 7SK ribonucleoprotein (RNP) complex that negatively regulates transcription elongation by RNA polymerase II

• Specifically binding to the highly conserved 3′-terminal U-rich stretch of 7SK RNA

• Regulating mRNA splicing fidelity by promoting U6 snRNA processing and 2'-O-methylation

• Supporting mitochondrial biogenesis and energy production through modulation of SIRT1 homeostasis and activity

• Maintaining proper cardiac function through regulation of oxidative phosphorylation

LARP7's activity spans multiple cellular processes, making it a subject of interest across various research disciplines including transcriptional regulation, RNA processing, and cardiac physiology.

What applications have been validated for LARP7 antibodies?

LARP7 antibodies have been successfully employed in multiple experimental techniques:

Each application requires specific optimization parameters, with Western blotting being the most extensively validated application across the literature.

What is the recommended protocol for Western blotting with LARP7 antibodies?

For optimal Western blotting results with LARP7 antibodies:

• Sample preparation: Extract proteins from cells or tissues using standard lysis buffers containing protease inhibitors.

• Protein separation: Separate 20-40 μg of protein lysate on an 8-12% SDS-PAGE gel.

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane.

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

• Primary antibody incubation: Dilute LARP7 antibody at 1:1000-1:4000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash 3-5 times with TBST.

• Secondary antibody incubation: Incubate with an appropriate HRP-conjugated secondary antibody at recommended dilution.

• Detection: Visualize using ECL substrate and imaging system.

The LARP7 protein appears at approximately 68-70 kDa on Western blots, with detection validated in multiple cell lines including Jurkat and NIH/3T3 cells, as well as various human tissues .

How can researchers investigate LARP7's role in transcriptional regulation?

To study LARP7's function in transcriptional regulation:

• Chromatin Immunoprecipitation (ChIP): Use LARP7 antibodies to identify genomic loci where 7SK RNP complexes associate with chromatin.

• RNA Immunoprecipitation (RIP): Apply LARP7 antibodies to identify RNA species interacting with LARP7, focusing on 7SK RNA and potential novel targets.

• Transcriptional reporter assays: Assess the impact of LARP7 depletion or overexpression on promoter activity using luciferase reporter systems. Research has shown that LARP7 knockdown enhances transcription from cellular polymerase II promoters and TAT-dependent HIV-1 promoters .

• Transcriptome analysis: Compare gene expression patterns between wild-type and LARP7-depleted cells to identify transcriptional networks regulated by LARP7.

These approaches should incorporate appropriate controls, including IgG controls for immunoprecipitation experiments and rescue experiments to confirm specificity of LARP7 depletion effects.

What methodological approaches are recommended for studying LARP7-7SK RNP complex dynamics?

To investigate LARP7's interactions within the 7SK RNP complex:

• Co-immunoprecipitation with LARP7 antibodies: Optimize protocols to preserve protein-RNA complexes by using mild lysis conditions and RNase inhibitors.

• RNA binding assays: Conduct in vitro transcription to generate 32P-labeled wild-type or mutant 7SK RNAs, then incubate with recombinant LARP7 protein before analysis on native polyacrylamide gels. Supershift experiments can be performed by adding 1 μg of LARP7 antibody .

• Drug treatment studies: Perform immunoprecipitations before and after treatment with transcriptional inhibitors like DRB (100 μM for 1 hour) to study dynamic changes in complex composition .

• RT-PCR analysis: Extract RNA from immunoprecipitates to confirm the presence of 7SK RNA and potentially identify other RNA species in LARP7 complexes .

These approaches reveal that LARP7 remains associated with 7SK RNA even after P-TEFb is released from the complex following stimulation .

How can researchers utilize LARP7 antibodies to study its role in cardiac function and mitochondrial biogenesis?

To investigate LARP7's role in cardiac function:

• Immunohistochemistry and immunofluorescence: Use LARP7 antibodies at 1:50-1:500 dilution on cardiac tissue samples from normal and diseased hearts to assess expression changes .

• Proximity ligation assays: Apply LARP7 antibodies in combination with SIRT1 antibodies to detect and quantify LARP7-SIRT1 interactions in situ in cardiac tissues.

• Mitochondrial analysis: Combine LARP7 immunostaining with mitochondrial markers to assess co-localization and potential direct interactions with mitochondrial components.

• In vivo models: Compare cardiac-specific LARP7 knockout mouse models with wild-type to examine defects in mitochondrial biogenesis, oxidative phosphorylation, and cardiac function. Research has demonstrated that cardiac-specific inactivation of LARP7 results in impaired mitochondrial biogenesis and heart failure by 4 months of age .

These approaches have revealed that LARP7 is essential for mitochondrial biogenesis and energy production through modulation of SIRT1 homeostasis, with LARP7 levels being significantly downregulated in failing human hearts .

What controls should be incorporated when using LARP7 antibodies in disease model studies?

When studying LARP7 in disease contexts:

• Antibody validation controls:

  • Include isotype control antibodies in all immunostaining experiments

  • Perform antibody validation using LARP7 knockout or knockdown models

  • Conduct peptide competition assays to confirm antibody specificity

• Experimental design controls:

  • Compare LARP7 expression across multiple disease models (e.g., different heart failure models)

  • Include time-course studies to track LARP7 changes during disease progression

  • Perform parallel analyses of known LARP7 interactors (e.g., components of 7SK RNP)

• Rescue experiments:

  • Restore LARP7 expression in disease models using adeno-associated virus (AAV) serotype 9-mediated gene delivery, which has been shown to improve function in injured hearts

  • Use small molecule inhibitors that affect LARP7 regulation (e.g., ATM inhibitors) as alternative approaches to rescue LARP7 function

These control strategies ensure that observed phenotypes are specifically related to LARP7 function rather than experimental artifacts or off-target effects.

How can researchers optimize immunoprecipitation protocols for studying LARP7-RNA interactions?

For successful LARP7-RNA interaction studies:

• Buffer optimization:

  • Use buffers containing 10 mM HEPES (pH 7.5), 10 mM KCl, 2 mM MgCl₂, 1 mM DTT, and 5% glycerol for binding assays

  • Include RNase inhibitors to prevent RNA degradation during sample processing

• Cross-linking considerations:

  • Employ UV cross-linking for direct RNA-protein interactions

  • Use formaldehyde for capturing larger ribonucleoprotein complexes

• Affinity purification:

  • For RNA-binding assays, incubate in vitro-transcribed 32P-labeled RNAs with 250 ng of recombinant LARP7 for 20 minutes at 20°C

  • Add 2 μg of heparin before loading onto native PAGE gels to reduce non-specific interactions

• Analysis methods:

  • Perform RT-PCR on immunoprecipitated RNAs to detect specific targets

  • Consider RNA sequencing of immunoprecipitated material to identify the complete repertoire of LARP7-associated RNAs

These optimized protocols have successfully demonstrated LARP7's specific binding to both 7SK RNA and U6 snRNAs .

How can LARP7 antibodies be used to investigate its role in cancer and Alazami syndrome?

LARP7 has been implicated in both cancer (characterized by hyperproliferation) and Alazami syndrome (characterized by growth retardation) . To investigate these connections:

• Cancer research applications:

  • Perform immunohistochemistry on cancer tissues using LARP7 antibodies at 1:50-1:500 dilution to assess expression patterns compared to normal tissues

  • Combine with proliferation markers to correlate LARP7 expression with cell proliferation status

  • Investigate mechanisms by which reduced LARP7 may contribute to enhanced transcription of oncogenes through deregulation of P-TEFb activity

• Alazami syndrome studies:

  • Analyze LARP7 expression, localization, and function in patient-derived cells using immunofluorescence (1:50-1:500 dilution)

  • Examine effects of LARP7 mutations on 7SK RNP complex assembly and function

  • Investigate developmental pathways affected by LARP7 dysfunction that may contribute to primordial dwarfism and intellectual disability

• Comparative approach:

  • Analyze how different mutations or expression levels of LARP7 can lead to opposite phenotypic outcomes (hyperproliferation versus growth retardation)

  • Study tissue-specific effects of LARP7 dysregulation

Understanding these seemingly contradictory roles may provide insights into the complex regulatory networks controlled by LARP7 in different cellular contexts.

What techniques can researchers use to study LARP7's role in oxidative stress response?

Given LARP7's connection to oxidative stress in heart failure pathogenesis :

• Oxidative stress induction models:

  • Treat cells with various oxidative stress inducers (H₂O₂, paraquat, etc.) and monitor LARP7 expression and localization using immunoblotting and immunofluorescence

  • Compare LARP7 levels before and after oxidative stress using Western blotting at 1:1000-1:4000 dilution

• Mechanism investigation:

  • Use LARP7 antibodies to study the ATM-mediated DNA damage response pathway activation following oxidative stress

  • Perform co-immunoprecipitation to detect increased LARP7 ubiquitination after oxidative stress exposure

  • Investigate whether antioxidant treatment prevents LARP7 degradation

• Functional consequences:

  • Examine how oxidative stress-induced LARP7 reduction affects SIRT1 stability and activity

  • Assess the impact on downstream targets of SIRT1, particularly genes involved in oxidative phosphorylation and energy metabolism

Research has revealed that elevated reactive oxygen species in failing hearts activate the ATM-mediated DNA damage response pathway, promoting LARP7 ubiquitination and degradation, which subsequently impairs SIRT1 function .