TBC1D7 Antibody

What is TBC1D7 and why is it important in cellular signaling research?

TBC1D7 is a protein that functions as the third core subunit of the tuberous sclerosis complex (TSC), specifically forming the TSC1-TSC2-TBC1D7 (TSC-TBC) complex. This complex plays a crucial role in regulating cell growth by inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) through its GTPase-activating protein (GAP) activity toward Rheb. TBC1D7's importance lies in its contribution to the functional complex that senses specific cellular growth conditions. Mutations in the TBC1D7 gene have been associated with increased mTORC1 signaling, delayed autophagy, intellectual disability (ID), and macrocrania, making it an important target for research into these conditions .

The study of TBC1D7 provides insights into growth regulation pathways and potential therapeutic targets for diseases associated with dysregulated mTORC1 signaling. Research has demonstrated that TBC1D7 is ubiquitously expressed across tissues, including those commonly affected in TSC disease, suggesting its broad physiological importance .

What types of TBC1D7 antibodies are available for research applications?

Based on the available information, researchers can access recombinant rabbit monoclonal antibodies against TBC1D7, such as the TBC1D7 (D8K1Y) Rabbit mAb #14949. This particular antibody has been validated for Western Blotting (WB) and Immunoprecipitation (IP) applications. The antibody demonstrates reactivity with human, mouse, and rat TBC1D7 proteins, making it versatile for studies across different model organisms .

The recombinant nature of this antibody provides superior lot-to-lot consistency, continuous supply, and animal-free manufacturing, which are important considerations for long-term research projects. The availability of well-characterized antibodies like this enables reliable detection of endogenous TBC1D7 in various experimental contexts .

How does TBC1D7 interact with the TSC1-TSC2 complex at the molecular level?

TBC1D7 interacts directly with the TSC1-TSC2 complex through binding to TSC1, specifically at the C-terminal coiled coil (CC) region of TSC1 (residues 939-992). Crystal structure analysis has revealed that two TSC1-CCs form a parallel homodimer, creating two symmetric surfaces for interaction with TBC1D7. The interaction occurs primarily through TBC1D7's α4 and α5 helices, which engage with the α1 helix of one TSC1 molecule through hydrophobic interactions, while simultaneously associating with the second TSC1 molecule via the C-terminal tip of its α4 helix .

Biochemical evidence indicates that TBC1D7 substantially stabilizes the homodimerization of TSC1-CC, contributing to the structural integrity of the complete TSC complex. Approximately 40-50% of total cellular TBC1D7 is associated with the TSC1-TSC2 complex, with the remainder existing as a free pool in cells. Importantly, while TBC1D7 can bind to TSC1 in the absence of TSC2, it primarily binds to the complete TSC1-TSC2 complex in wild-type cells .

What are the recommended protocols for using TBC1D7 antibodies in Western Blotting?

For Western Blotting applications using TBC1D7 antibodies such as the D8K1Y Rabbit mAb, a dilution of 1:1000 is recommended based on manufacturer guidelines. The molecular weight of TBC1D7 is approximately 30 kDa, which should be considered when analyzing Western blot results. To ensure specificity, proper controls should be included, such as lysates from cells with siRNA-mediated knockdown of TBC1D7 .

When designing Western blot experiments to study TBC1D7, researchers should consider the stability of the protein. The unbound pool of TBC1D7 turns over rapidly compared to the portion bound to the TSC1-TSC2 complex. Therefore, experimental conditions that might affect TSC1 or TSC2 levels can indirectly affect TBC1D7 stability and detection. When TSC1 is depleted, both TBC1D7 and TSC2 show substantially increased rates of degradation, indicating that TSC1 stabilizes both proteins .

How can researchers effectively use TBC1D7 antibodies for immunoprecipitation studies?

To validate the specificity of TBC1D7 antibodies for immunoprecipitation, controls should include siRNA-mediated knockdown of TBC1D7. For studying complex formation, reciprocal immunoprecipitations with antibodies to TSC1 or TSC2 can confirm the endogenous interaction of TBC1D7 with the TSC1-TSC2 complex. This approach has been successfully used across a diverse series of mouse tissues, indicating the ubiquitous nature of this interaction .

What experimental approaches can be used to study the proportion of TBC1D7 associated with the TSC1-TSC2 complex?

To determine what proportion of total cellular TBC1D7 is associated with the TSC1-TSC2 complex, researchers can employ sucrose density gradient fractionation followed by Western blotting. This approach has revealed that TBC1D7 fractionates into two distinct pools: one in low-density fractions (free TBC1D7) and a second in high-density fractions that also contain TSC1 and TSC2 (complex-associated TBC1D7) .

Co-immunoprecipitation experiments from these fractions can confirm that TBC1D7 co-immunoprecipitates with TSC2 only from the denser fractions, verifying that their co-fractionation reflects their association. Through such experiments, researchers have determined that approximately 40-50% of total cellular TBC1D7 is associated with the TSC1-TSC2 complex. This methodology can also reveal that TSC2 exists as both TSC1-free and TSC1-associated entities in cells, providing insights into complex dynamics .

How does TBC1D7 knockdown affect the stability and function of the TSC1-TSC2 complex?

When TBC1D7 is knocked down using siRNA or shRNA techniques, the TSC1-TSC2 complex still forms, but there is a reproducible reduction in the association between TSC1 and TSC2. This can be demonstrated through co-immunoprecipitation experiments and sucrose density fractionation studies. Specifically, TBC1D7 knockdown results in the shifting of some TSC1 and TSC2 to intermediate-density fractions containing the uncomplexed proteins, consistent with partial dissociation of the complex .

What structural insights can inform the design of experiments targeting the TBC1D7-TSC1 interaction?

Crystal structure analysis of the TBC1D7-TSC1 complex provides valuable insights for designing experiments targeting this interaction. The structure reveals that TBC1D7 binds to a TSC1 homodimer, with each TBC1D7 molecule interacting with both TSC1 molecules in the dimer. Specifically, TBC1D7 employs its α4 and α5 helices to interact with the α1 helix of one TSC1 molecule mainly through hydrophobic interactions, while simultaneously associating with the second TSC1 molecule using the C-terminal tip of its α4 helix .

Researchers designing mutagenesis experiments to disrupt this interaction should focus on critical interface residues identified in the crystal structure. Mutations to these residues have been shown to greatly compromise TBC1D7's ability to stabilize TSC1 dimerization. Additionally, understanding that TBC1D7 stabilizes TSC1 homodimerization provides a mechanistic explanation for how TBC1D7 contributes to the structural integrity of the complete TSC complex .

How can researchers differentiate between free and complex-associated pools of TBC1D7 in cells?

To differentiate between free and complex-associated pools of TBC1D7 in cells, researchers can employ a combination of biochemical fractionation and stability assays. Sucrose density gradient fractionation separates TBC1D7 into two pools: a low-density free pool and a high-density complex-associated pool that co-fractionates with TSC1 and TSC2 .

The stability of these two pools can be distinguished through cycloheximide chase assays, which have demonstrated that only the free pool of TBC1D7 (in low-density fractions) is rapidly degraded over a 3-hour treatment, while the complex-associated pool remains stable. This differential stability provides another approach to distinguish between the two pools. Additionally, knockdown of TSC1 causes a complete shift of TBC1D7 to low-density fractions, confirming that TBC1D7 resides in high-density fractions solely due to its association with the TSC1-TSC2 complex .

What controls should be included when validating TBC1D7 antibody specificity?

When validating TBC1D7 antibody specificity, researchers should include several key controls. First, siRNA-mediated knockdown of TBC1D7 should be performed to confirm the specificity of the antibody signal in Western blots and immunoprecipitation experiments. This approach has been successfully used to validate antibodies against endogenous TBC1D7 .

For immunofluorescence studies, extensive validation is necessary as many commercially available antibodies may not show specific localization patterns. Researchers should test antibodies by comparing staining patterns between control and TBC1D7-knockdown cells. It is worth noting that while specific antibodies for TSC2 immunofluorescence have been identified (showing a punctate localization pattern throughout the cell with concentration in perinuclear regions), to date, antibodies that reliably reveal specific localization patterns for endogenous TBC1D7 have been challenging to identify .

How can researchers investigate the role of TBC1D7 in mTORC1 signaling regulation?

To investigate TBC1D7's role in mTORC1 signaling regulation, researchers can employ both loss-of-function and structural studies. siRNA or shRNA-mediated knockdown of TBC1D7 can be used to assess its contribution to TSC1-TSC2 complex stability and subsequent effects on mTORC1 activity. Key readouts of mTORC1 activity include phosphorylation of downstream targets such as S6K1 and 4E-BP1, which can be measured by Western blotting with phospho-specific antibodies .

For a more comprehensive understanding, researchers can combine TBC1D7 knockdown with various cellular stresses known to inhibit mTORC1 (such as amino acid starvation, glucose deprivation, or growth factor withdrawal) to determine whether TBC1D7 is required for specific stress responses. Additionally, structure-guided mutagenesis of key residues at the TBC1D7-TSC1 interface can help elucidate how the molecular interaction contributes to mTORC1 regulation without completely eliminating TBC1D7 expression .

What technical considerations are important when studying TBC1D7 in different experimental models?

When studying TBC1D7 in different experimental models, researchers should consider several technical aspects. First, the TBC1D7 (D8K1Y) Rabbit mAb has been validated for reactivity with human, mouse, and rat TBC1D7, making it suitable for studies across these species. This is particularly valuable for translational research bridging findings between animal models and human samples .

For disease-related studies, it's important to note that mutations in TBC1D7 have been associated with intellectual disability and macrocrania, while also leading to increased mTORC1 signaling and delayed autophagy. Researchers studying these conditions should consider including analysis of autophagy markers alongside mTORC1 signaling readouts when investigating TBC1D7 function .