GLI2 Antibody

What is GLI2 and what cellular functions does it regulate?

GLI2 belongs to the GLI C2H2-type zinc-finger protein family and functions as a biopotential transcription regulator in the Hedgehog signaling pathway. It contains 5 conserved tandem C2H2 zinc finger domains that enable DNA binding to the sequence 5'-GAACCACCCA-3', which forms part of the TRE-2S regulatory element . This transcription factor plays essential roles during embryogenesis and activates patched Drosophila homolog (PTCH) gene expression . GLI2 possesses both repression domains in its N-terminal region and activation domains in its C-terminal portion, allowing for complex transcriptional regulation . The dysregulation of GLI2 is associated with several developmental disorders including Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome, and various forms of polydactyly .

What applications are GLI2 antibodies validated for?

GLI2 antibodies have been extensively validated across multiple experimental applications as summarized in the table below:

It is essential to optimize these dilutions for each specific experimental system to obtain optimal results, as antibody performance can be sample-dependent .

What are the validated sample types for GLI2 antibody reactivity?

GLI2 antibodies have demonstrated reliable detection across various biological samples:

For developmental studies, GLI2 has been successfully detected in mouse embryo sections, specifically in developing muscle tissue using immunohistochemistry techniques .

What is the molecular weight of GLI2 protein and how does it appear on Western blots?

The molecular characterization of GLI2 reveals interesting differences between calculated and observed weights:

These variations reflect the complex post-translational processing of GLI2. When performing Western blots, researchers should anticipate detecting both the full-length protein and potentially its processed forms. The appearance of multiple bands is consistent with the protein's known processing mechanisms and should not necessarily be interpreted as non-specific binding .

What are the recommended storage conditions for GLI2 antibodies?

Proper storage is crucial for maintaining antibody performance:

Following manufacturer-specific storage recommendations ensures optimal antibody performance throughout the expected shelf life.

How is GLI2 processed and regulated at the post-translational level?

GLI2 post-translational regulation is complex and differs significantly from other GLI family members:

Unlike GLI3 and Cubitus Interruptus (the fly homolog), only a minor fraction of GLI2 is proteolytically processed to form a transcriptional repressor in vivo . The full-length GLI2 protein is primarily regulated through degradation rather than processing . This degradation pathway involves a sophisticated phosphorylation cascade:

• Initial phosphorylation by protein kinase A (PKA) at a cluster of serine residues in the carboxyl terminus

• Subsequent phosphorylation by casein kinase 1 (CK1)

• Further phosphorylation by glycogen synthase kinase 3 (GSK3)

• Direct interaction of phosphorylated GLI2 with βTrCP in the SCF ubiquitin-ligase complex

• Ubiquitination of GLI2 leading to proteasomal degradation

Importantly, both the processing and degradation mechanisms are suppressed by active Sonic hedgehog (Shh) signaling in vivo, providing a molecular basis for GLI2 activation in response to Hedgehog pathway stimulation .

What methodological approaches enable detection of GLI2 in difficult samples?

For challenging samples, several technical modifications can improve GLI2 detection:

For immunohistochemistry applications, antigen retrieval is critical and should be performed with TE buffer pH 9.0, though citrate buffer pH 6.0 can serve as an alternative . When working with embryonic or developmental tissues, specific fixation protocols are essential - as demonstrated in mouse embryo studies where GLI2 was successfully visualized in developing muscle using affinity-purified antibodies and HRP-DAB detection systems counterstained with hematoxylin .

For protein extractions intended for Western blotting, inclusion of proteasome inhibitors may enhance detection of full-length GLI2 by preventing its rapid degradation . Given the molecular weight of GLI2 (>160 kDa), extended gel run times and optimal transfer conditions are necessary for proper resolution and detection.

How does Sonic hedgehog signaling regulate GLI2 transcriptional activity?

Sonic hedgehog (Shh) signaling exerts precise control over GLI2 function through multiple mechanisms:

The transcriptional activity of GLI2 is primarily regulated through a suppression of both its proteolytic processing and degradation . In the absence of Shh signaling, GLI2 undergoes the phosphorylation cascade described above, leading to its ubiquitination and proteasomal degradation . This process limits the amount of active, full-length GLI2 available for transcriptional activation.

When Shh signaling is active, this phosphorylation and subsequent degradation is inhibited, allowing full-length GLI2 to accumulate and function as a transcriptional activator . This regulatory mechanism differs from GLI3, which is predominantly regulated through proteolytic processing rather than degradation. The molecular basis for this difference in regulation between GLI family members remains an active area of research.

What role does GLI2 play in T-cell receptor signaling and immune regulation?

Recent research has revealed GLI2's unexpected role in T-cell biology:

Experimental evidence demonstrates that GLI2 modulates T-cell receptor signaling and affects interleukin-2 (IL-2) production . Studies comparing wild-type cells with Gli2A (activator) cells showed that fewer CD4+ Gli2A cells expressed intracellular IL-2 . This effect was proven to be cell-intrinsic through isolated CD4+ T-cell cultures with anti-CD3/CD28 stimulation.

The impact on IL-2 signaling was particularly pronounced in IL-2hi CD25+ cells, which were significantly diminished in Gli2A cultures compared to wild-type at both 48 and 72 hours . Quantitative analysis of culture supernatants confirmed lower IL-2 protein levels in Gli2A conditions, with the most statistically significant difference occurring at 48 hours post-stimulation .

Functionally, this translates to proliferation defects: while wild-type cells showed enhanced proliferation when IL-2 was added together with anti-CD3/CD28 stimulation (approximately threefold more cells undergoing multiple divisions), Gli2A cells failed to demonstrate this proliferative advantage . This ultimately resulted in lower CD4+ cell numbers in Gli2A cultures at 72 hours, even in the presence of exogenous IL-2 .

How can researchers validate GLI2 antibody specificity in experimental systems?

Rigorous validation is essential for ensuring reliable research outcomes:

Multiple approaches should be employed for comprehensive validation:

• Genetic validation: Compare antibody reactivity in wild-type versus GLI2 knockout or knockdown models. The search results indicate that at least 4 publications have used GLI2 antibodies in knockout/knockdown studies .

• Multiple application validation: Test the antibody across several applications (WB, IHC, IF) to confirm consistent detection patterns. The GLI2 antibody from Proteintech (18989-1-AP) has been validated in 47+ Western blot studies, 9+ IHC applications, and 10+ immunofluorescence experiments .

• Cross-species validation: Confirm specificity across relevant species. The GLI2 antibodies described have demonstrated reactivity with human, mouse, and rat samples, with some predicted to work in additional species like monkey and pig .

• Multiple epitope targeting: Consider using antibodies targeting different regions of GLI2 to validate observations, especially when studying processing or isoform expression.

• Proper controls: Always include negative controls (isotype controls, secondary antibody-only controls) as demonstrated in the mouse embryo staining protocol where lack of labeling was confirmed when primary antibodies were omitted .

What are the key considerations when studying GLI2 in developmental contexts?

Developmental research presents unique challenges for GLI2 investigation:

GLI2 plays crucial roles during embryogenesis and has been implicated in multiple developmental disorders . When studying GLI2 in embryonic or developmental tissues, researchers should consider temporal expression patterns, as GLI2 expression and activity may change throughout developmental stages.

Spatial expression is equally important - GLI2 detection in mouse embryo (15 d.p.c.) revealed specific localization to developing muscle tissue . This highlights the importance of proper sectioning and anatomical characterization when performing developmental studies.

The interaction between GLI2 and other developmental signaling pathways (beyond Hedgehog) should also be considered, as cross-talk between pathways may influence GLI2 expression and function in tissue-specific ways.

How can GLI2 processing variants be distinguished experimentally?

Differentiating between GLI2 variants requires specialized approaches:

To differentiate between full-length GLI2 (~167-185 kDa) and its processed form (~88 kDa), researchers should:

• Select antibodies with epitopes that can distinguish between forms - N-terminal antibodies will detect both full-length and processed forms, while C-terminal antibodies will detect only the full-length protein

• Optimize gel systems to properly resolve high molecular weight proteins, using gradient gels or extended run times if necessary

• Consider using phospho-specific antibodies to detect the phosphorylated forms of GLI2 that precede degradation

• Implement experimental manipulations of the Hedgehog pathway to alter the balance between GLI2 forms, as Shh signaling suppresses both processing and degradation

• Use proteasome inhibitors to prevent degradation and capture the various processing intermediates