ARHGAP19 Antibody

What is ARHGAP19 and what cellular functions does it regulate?

ARHGAP19 (Rho GTPase-activating protein 19) belongs to the ARHGAP family that encodes negative regulators of Rho GTPases. As a GAP-domain containing protein, it specifically stimulates GTP hydrolysis by RhoA, but not Rac1 or CDC42 . ARHGAP19 is involved in cell migration, proliferation, differentiation, actin remodeling, and G1 cell cycle progression . Most significantly, it plays essential roles in the proper division of T lymphocytes by controlling cytokinesis and chromosome segregation .

What is the expression pattern of ARHGAP19 across tissue types?

ARHGAP19 demonstrates a predominantly hematopoietic expression pattern. It is highly expressed in blood cells and their precursors, with particularly strong expression detected in lymphocyte cell lines including HL-60, Jurkat, and K-562 cells . According to supplementary research, with the notable exception of germ cells, ARHGAP19 expression outside the hematopoietic system is relatively low . This restricted expression pattern suggests specialized functions in blood cell biology.

What are the recommended applications and dilutions for ARHGAP19 antibodies?

Based on validated research protocols, ARHGAP19 antibodies are optimized for the following applications:

Researchers should note that optimal dilutions may be sample-dependent, and titration is recommended for each specific experimental system to obtain optimal results .

How does ARHGAP19 regulate mitotic processes in lymphocytes?

Time-lapse microscopy experiments have revealed that ARHGAP19 plays a dual role in lymphocyte division by regulating:

• Cell shape dynamics: ARHGAP19 controls the precise timing of cell elongation and cleavage furrow formation. In control cells, elongation and furrow ingression occur approximately 3 and 4 minutes after anaphase onset, respectively. Overexpression of ARHGAP19 delays these events to 7 and 8 minutes post-anaphase onset. Conversely, ARHGAP19-depleted cells begin elongation prematurely (approximately 2 minutes before anaphase onset) with furrow ingression occurring just 2 minutes after anaphase onset .

• Chromosomal integrity: Approximately 44% of ARHGAP19-deficient lymphocytes exhibit severe chromosome mis-segregation, often preceded by extensive cell shape remodeling in early mitosis . These findings indicate ARHGAP19's critical role in maintaining chromosomal stability during cell division.

These regulatory functions are dependent on ARHGAP19's GAP activity, as mutation of the catalytic arginine residue (R143) in the GAP domain results in phenotypes opposite to those of the wild-type protein .

What is the biochemical mechanism of ARHGAP19's GAP activity?

ARHGAP19 functions as a specific GAP for RhoA. In vitro GAP assays demonstrate that wild-type ARHGAP19 stimulates GTP hydrolysis catalyzed by RhoA, but shows no activity toward Rac1 or CDC42 . The R143A mutant of ARHGAP19, with a mutation in the critical catalytic arginine residue, displays no GAP activity, confirming the specificity of this catalytic mechanism .

In vivo studies with a cytoplasmic mutant of GFP-ARHGAP19 (truncated of its C-terminal region containing nuclear localization signals) show decreased cellular levels of active RhoA but not Rac1 or CDC42. This mutation also decreases stress fiber formation in interphase cells and impairs RhoA-dependent cell rounding that normally occurs at the beginning of mitosis in adherent cells - all effects dependent on arginine 143 of the GAP domain .

How do mutations in ARHGAP19's GAP domain affect cellular phenotypes?

Mutation of the catalytic arginine residue (R143) in ARHGAP19's GAP domain produces significant phenotypic alterations:

• Mitotic abnormalities: Approximately 65.7% of cells expressing the R143A mutant exhibit mitotic defects, including premature ovoid cell morphology at metaphase and defective chromosome segregation .

• Cortical hypercontractility: The mutant induces major membrane blebbing and/or formation of additional cleavage furrows. Post-cytokinesis, daughter cells often demonstrate excessive movement for several hours .

• Protein recruitment effects: Expression of the R143A mutant enhances membrane recruitment of RhoA, citron, and myosin IIA in approximately 50% of cells during prophase and metaphase stages .

These findings indicate that the R143A mutant acts as a dominant negative, producing effects similar to ARHGAP19 silencing, which confirms that the wild-type protein's functions are dependent on an intact GAP domain .

How does ARHGAP19 interact with the cellular cytoskeleton?

ARHGAP19 regulates cytoskeletal dynamics through several interconnected mechanisms:

• RhoA pathway modulation: As a GAP for RhoA, ARHGAP19 controls actin cytoskeleton remodeling. Expression of ARHGAP19 decreases stress fiber formation in interphase cells - a process known to be downstream of RhoA activity .

• Protein recruitment regulation: ARHGAP19 controls the recruitment of critical cytoskeletal regulators, including citron and myosin IIA, to the plasma membrane of mitotic lymphocytes. Silencing of ARHGAP19 enhances membrane recruitment of these proteins in approximately 50% of cells during prophase and metaphase .

• Intermediate filament organization: ARHGAP19 regulates the vimentin intermediate filament network through control of Rock2-mediated phosphorylation. This vimentin network forms a critical cage-like structure in lymphocytes that maintains cellular stiffness and shape during mitosis .

What is the relationship between ARHGAP19 and Rock2-mediated phosphorylation?

ARHGAP19 exerts precise control over Rock2-mediated phosphorylation events in mitotic cells:

• Regulation of Rock2 activation: In control lymphocytes, active Rock2 (phospho-S1366) localizes to a distinctive cage-like structure during prophase and metaphase. Overexpression of wild-type ARHGAP19 dramatically reduces active Rock2 levels during these phases .

• Vimentin phosphorylation control: Active Rock2 colocalizes with phosphorylated vimentin (pS71-vimentin) in lymphocytes. ARHGAP19 regulates vimentin phosphorylation at S71 through its control of Rock2 activity, with levels of pS71-vimentin correlating with active Rock2 levels throughout mitosis progression .

• Functional significance: The vimentin intermediate filament network is crucial for maintaining lymphocyte rigidity and morphology. Disruption of Rock2-mediated vimentin phosphorylation through modulation of ARHGAP19 levels affects cell shape changes during mitosis, impacting the timing of cell elongation, cleavage furrow formation, and chromosome segregation .

What is the emerging significance of ARHGAP19 in cancer research?

ARHGAP19 and related ARHGAP family members demonstrate potential significance in cancer biology:

• Hematological malignancies: ARHGAP19 expression has been detected in human T-cell acute lymphoblastic leukemia samples. Interestingly, it clusters with genes involved in cell division, including Ect2, RacGAP1, and citron (proteins with established roles in cytokinesis), suggesting a potential role in leukemic cell proliferation .

• Tumor microenvironment modulation: ARHGAP family genes correlate with a tumor-promoting microenvironment characterized by lower Th1/Th2 cell ratios, higher dendritic cell infiltration, and increased regulatory T cell presence .

• Bladder cancer biomarker potential: Members of the ARHGAP family have been identified as potential novel biomarkers in bladder cancer, with expression levels potentially correlating with patient prognosis .

What experimental systems are available for modulating ARHGAP19 expression?

Researchers have employed several approaches to manipulate ARHGAP19 expression:

• Overexpression systems:

  • Doxycycline-inducible expression of GFP-ARHGAP19 fusion proteins

  • Expression of domain-specific mutants (e.g., R143A GAP-deficient mutant)

  • Truncated forms lacking specific domains (e.g., ΔCter mutant without C-terminal nuclear localization signals)

• Silencing strategies:

  • shRNA constructs targeting ARHGAP19 mRNA

  • Recombinant plasmid vectors delivered via viral transduction systems

• Cell models:

  • Kit225 lymphocytes: Primary model for studying ARHGAP19 function in hematopoietic cells

  • HeLa cells: System with minimal endogenous ARHGAP19 expression for studying cytoplasmic mutants

  • Bladder cancer lines (T24 and UM-UC-3): Models for oncology research

• Selection methods:

  • Puromycin selection (1.0 μg/ml) for isolating cells with stable integration

What controls should be implemented when studying ARHGAP19?

Robust experimental design for ARHGAP19 research requires several key controls:

• Expression controls:

  • Positive controls: Hematopoietic cell lines with known ARHGAP19 expression (HL-60, Jurkat, K-562)

  • Negative controls: Cell types with minimal ARHGAP19 expression (most non-hematopoietic cells)

• Functional GAP activity controls:

  • Wild-type ARHGAP19: Positive control for GAP activity

  • R143A mutant: Negative control lacking GAP activity

  • Comparative analysis with different GTPases (RhoA, Rac1, CDC42) to demonstrate specificity

• Expression modulation controls:

  • Both silencing and overexpression systems should be employed

  • Vector controls (empty vector or non-targeting shRNA) to account for non-specific effects

  • Validation of expression changes via western blot analysis

• Mitotic timing controls:

  • Time-lapse microscopy to precisely determine key mitotic events

  • Careful documentation of anaphase onset and other critical timepoints

  • Comparative analysis across different mitotic phases

How can researchers troubleshoot ARHGAP19 antibody applications?

When optimizing ARHGAP19 antibody applications, researchers should consider:

• Western blot optimization:

  • Use recommended dilutions (1:1000-1:6000) as starting points

  • Expected molecular weight of approximately 54 kDa

  • Sample-dependent results may require protocol adjustments

• Immunohistochemistry considerations:

  • Analyze multiple high-power fields (minimum of three randomized fields)

  • Quantitate positive-staining cells and use mean values for analysis

  • For low-abundance markers, consider whole-section quantitation

• Subcellular localization awareness:

  • Native ARHGAP19 contains bipartite nuclear localization signals in its C-terminal region

  • Wild-type protein may demonstrate nuclear localization

  • Truncated forms lacking the C-terminal region typically show cytoplasmic distribution

• Antibody validation:

  • Verify antibody specificity through immunoprecipitation followed by western blot

  • For co-localization studies, confirm that antibodies do not cross-react

  • Include appropriate negative controls to assess non-specific binding