arhgap42a Antibody

What is ARHGAP42 and what are its primary functions?

ARHGAP42 is a Rho GTPase-activating protein (RhoGAP) that selectively inhibits RhoA activity in vascular smooth muscle cells. It belongs to the GRAF (GAP for Rho associated with focal adhesion kinase) family of RhoGAPs . ARHGAP42 primarily functions as:

• A regulator of blood pressure by inhibiting RhoA-dependent contractility in vascular smooth muscle

• A component of focal adhesions and stress fibers, suggesting a role in cytoskeletal organization

• A potential modulator of cell migration and invasion in certain contexts

The protein contains N-terminal tandem Bin/amphiphysin/Rvs (BAR) and pleckstrin homology (PH) domains, with GAP activity that is autoinhibited by its BAR domain . Importantly, ARHGAP42 shows GAP activity toward RhoA and Cdc42, but not Rac1 .

ARHGAP42 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Most antibodies are tested at dilutions ranging from 1:1000 to 1:2000, with the predicted band size of approximately 99 kDa

• Immunohistochemistry on Paraffin sections (IHC-P): Typically used at dilutions of 1:50 to 1:200

• Immunoprecipitation (IP): Phospho-specific antibodies have been validated for IP at 1:100 dilution

• ELISA: Multiple antibodies are validated for ELISA applications

Examples of validated tissues include human placenta lysate for WB and paraffin-embedded human brain and testis tissues for IHC-P .

How can ARHGAP42 antibodies be utilized to study blood pressure regulation mechanisms?

ARHGAP42 has been identified as a critical regulator of blood pressure through its inhibition of RhoA-dependent contractility in vascular smooth muscle cells. To investigate this mechanism:

• Genotype-phenotype correlation studies: Use ARHGAP42 antibodies in combination with SNP genotyping (particularly rs604723) to correlate protein expression with blood pressure phenotypes

• Mechanotransduction experiments: Since ARHGAP42 expression is upregulated by cell stretch and tension in a RhoA-dependent manner, use antibodies to detect changes in expression under different mechanical conditions:

  • Apply cyclic stretch to SMC cultures using Flexcell systems

  • Pretreat with ROCK inhibitors like Y-27632 to determine pathway specificity

  • Measure ARHGAP42 expression changes in response to sphingosine 1-phosphate (a strong activator of RhoA signaling)

• Hypertension models: In DOCA-salt hypertension mouse models, ARHGAP42 antibodies can be used to monitor expression changes during disease progression

Research has shown that minor T allele at rs604723 increases ARHGAP42 expression approximately 3-fold in aorta and coronary artery samples compared to the major C allele, correlating with reduced blood pressure .

What are optimal methods for detecting ARHGAP42 phosphorylation and its functional significance?

ARHGAP42 is activated by Src-mediated tyrosine phosphorylation, particularly at tyrosine 376 (Tyr-376), which stimulates its GAP activity:

• Phospho-specific antibody application: Use phospho-ARHGAP42 (Tyr376) antibodies for:

  • Western blotting at 1:1000 dilution

  • Immunoprecipitation at 1:100 dilution

• Experimental design for phosphorylation studies:

  • Co-express ARHGAP42 with constitutively active Src (Src-F529)

  • Compare wild-type ARHGAP42 with Y376F mutant (phospho-deficient)

  • Immunoprecipitate with anti-GFP antibody (for tagged constructs)

  • Immunoblot with either anti-pTyr antibody or phospho-specific pY376 antibody

Functional studies have demonstrated that Src-mediated phosphorylation of ARHGAP42 Tyr-376 promotes focal adhesion dynamics and cell motility by stimulating GAP activity .

How can researchers validate ARHGAP42 antibody specificity in their experimental systems?

To ensure experimental validity, researchers should perform comprehensive antibody validation:

• Genetic validation:

  • Use CRISPR/Cas9-mediated gene editing to delete ARHGAP42 in cell lines

  • Design guide RNAs flanking conserved regions (e.g., DHS2 region)

  • Transfect cells with CRISPR constructs and verify deletion by PCR

  • Compare antibody reactivity between wild-type and knockout samples

• Expression modulation controls:

  • Knockdown ARHGAP42 using siRNA

  • Overexpress tagged ARHGAP42 constructs

  • Verify antibody signal changes accordingly in Western blot or immunostaining

• Allele-specific validation:

  • For studying genetic variants, design PCR primers that can distinguish between alleles (e.g., major C and minor T alleles at rs604723)

  • Perform antibody validation in heterozygous cell lines where expression differences can be measured

• Domain-specific controls:

  • Express truncated ARHGAP42 constructs lacking specific domains

  • Verify antibody reactivity with the appropriate domain-containing constructs

What considerations should be taken when studying ARHGAP42 subcellular localization?

ARHGAP42 localizes to both focal adhesions and actin stress fibers, requiring specific experimental approaches:

• Immunofluorescence optimization:

  • Fix cells using paraformaldehyde to preserve cytoskeletal structures

  • Permeabilize with mild detergents to maintain focal adhesion integrity

  • Co-stain with focal adhesion markers (e.g., paxillin, vinculin) and F-actin markers (phalloidin)

  • Use GFP-tagged ARHGAP42 constructs as positive controls

• Domain requirements for localization:

  • The SH3 domain of ARHGAP42 is indispensable for targeting to both actin stress fibers and focal adhesions

  • Use domain deletion constructs to verify specificity of antibody staining patterns

• Dynamic localization studies:

  • Monitor localization changes during cell adhesion, spreading, and migration

  • Combine with phospho-specific antibodies to correlate activation state with localization

How can researchers investigate ARHGAP42 genetic variants using antibody-based approaches?

ARHGAP42 genetic variants, particularly rs604723, significantly affect expression levels and are associated with blood pressure regulation:

• Allele-specific expression analysis:

  • Design PCR primers that distinguish between the major C and minor T alleles at rs604723

  • Quantify mRNA levels in heterozygous human aortic SMC cultures

  • Use DNase treatment and appropriate controls to eliminate genomic DNA contamination

  • Compare expression levels between alleles

• Regulatory element identification:

  • Use CRISPR/Cas9-mediated deletion of regulatory elements (such as DHS2)

  • Measure changes in endogenous ARHGAP42 expression

  • Combine with ChIP assays to identify transcription factor binding (e.g., serum response factor)

• Tissue-specific expression patterns:

  • Analyze expression in different vascular beds and tissues using IHC

  • Compare with GTEx data that shows approximately 3-fold higher ARHGAP42 mRNA levels in aorta and coronary artery samples from individuals homozygous for the minor allele

A well-characterized cohort study of 346 borderline hypertensive patients revealed an age-independent decrease in blood pressure in subjects homozygous for the minor allele compared with subjects homozygous for the major allele (76.9 mmHg vs. 81.8 mmHg, p = 0.028) .

What protocols are recommended for effective Western blot detection of ARHGAP42?

For optimal Western blot results with ARHGAP42 antibodies:

• Sample preparation:

  • Use RIPA or NP-40 based lysis buffers with protease and phosphatase inhibitors

  • Include phosphatase inhibitors particularly when studying phosphorylated forms

  • Sonicate lysates briefly to shear DNA and reduce viscosity

• Electrophoresis and transfer parameters:

  • Use 7.5% or 8% SDS-PAGE gels to resolve the 99-125 kDa ARHGAP42 protein effectively

  • Transfer to PVDF membranes at lower voltage for longer periods (e.g., 30V overnight)

• Antibody incubation:

  • Block with 5% BSA in TBST

  • Use primary antibody at 1:1000 to 1:2000 dilution

  • Incubate with secondary antibody (anti-rabbit) at 1:50000 dilution

• Expected results:

  • Predicted band size: 99 kDa

  • Observed band size in human placenta lysate: 99 kDa

What are the optimal methods for quantitative PCR analysis of ARHGAP42 expression?

To accurately measure ARHGAP42 expression by qPCR:

• RNA isolation and quality control:

  • Use RNeasy Mini Kit or similar high-quality RNA isolation methods

  • Treat with DNase to eliminate genomic DNA contamination

  • Verify RNA integrity by gel electrophoresis or Bioanalyzer

• cDNA synthesis:

  • Use iScript cDNA synthesis kit or similar reverse transcription systems

  • Include controls without reverse transcriptase to detect genomic contamination

• Primer design for standard qPCR:

  • Human ARHGAP42 exons 2-4: 5′-TTGGAGATGCAGAAACTGATGA-3′ and 5′-TTTGAATCAGTCTACGCCTTTCTTC-3′

  • Rat Arhgap42: 5′-TTCTGCATCTCCGATACAGTC-3′ and 5′-ATCAAAGAGCTGCTGAAGGATG-3′

  • GAPDH (control): 5′-ATGGGTGTGAACCACGAGAA-3′ and 5′-GGCATGGACTGTGGTCATGA-3′

• Allele-specific PCR primers for rs604723 variation:

  • T allele: 5′-TGTTGTTCCAAGGGTTCTT-3′

  • C allele: 5′-TGTTGTTCCAAGGGTTCTC-3′

• Experimental design:

  • Use 20 ng cDNA for each qPCR reaction

  • Include technical triplicates for each sample

  • Use reference genes appropriate for the tissue type being studied

How can ARHGAP42 antibodies be used to investigate disease mechanisms?

ARHGAP42 has been implicated in multiple disease contexts that can be investigated using antibodies:

• Hypertension studies:

  • Compare ARHGAP42 expression in normotensive versus hypertensive tissues

  • Correlate with genotype at rs604723 or other SNPs

  • Study in animal models of hypertension (e.g., DOCA-salt)

• Cancer research applications:

  • Investigate ARHGAP42 in nasopharyngeal carcinoma and other cancer types

  • Examine involvement in PI3K/Akt signaling pathways

  • Study the role in cell migration and invasion

• Mechanistic studies:

  • Investigate phosphorylation status using phospho-specific antibodies

  • Correlate with activation of Src family kinases

  • Study colocalization with focal adhesion components