CYCT1-2 Antibody

What are the molecular characteristics of CYCT1 and CYCT2 proteins?

CYCT1 (Cyclin T1) is a regulatory protein with a canonical amino acid length of 726 residues and a molecular weight of approximately 80.7 kDa, though the observed molecular weight in Western blotting is typically around 81 kDa . The protein is primarily localized in the nucleus and is widely expressed across multiple tissue types. CYCT1 is encoded by the CCNT1 gene (Gene ID: 904) .

CYCT2 is a related cyclin protein that also forms part of the P-TEFb complex. Research has established that CYCT1 and CYCT2 are distinct proteins that regulate different subsets of genes, as demonstrated by genetic knockout studies showing that CYCT2-deficient mice display embryonic lethality, indicating non-redundant functions with CYCT1 .

What applications are CYCT1 and CYCT2 antibodies validated for?

CYCT1 antibodies are validated for multiple research applications, with specific dilution recommendations varying by application :

When selecting an antibody, researchers should validate application-specific performance in their experimental system, as optimal dilutions may vary based on sample type and detection method .

How do CYCT1 and CYCT2 function in cellular processes?

CYCT1 and CYCT2 are regulatory subunits of the cyclin-dependent kinase pair complexes with CDK9, collectively forming the positive transcription elongation factor b (P-TEFb) . This complex plays a critical role in transcriptional elongation by phosphorylating the C-terminal domain of RNA polymerase II, facilitating productive transcription elongation .

CYCT1 specifically serves as an essential cofactor for HIV Tat protein, promoting RNA Pol II activation and allowing transcription of viral genes . In the context of HIV infection, CYCT1 binds to the transactivation domain of viral nuclear transcriptional activator Tat, enhancing transcriptional activation .

Research using genetic knockout models has demonstrated that CYCT1 and CYCT2 regulate distinct subsets of genes important for embryonic development and are not functionally redundant . In C. elegans, simultaneous inactivation of both cyclins produced a phenotype similar to RNA polymerase II inactivation, while individual inactivation had no effect, suggesting some functional overlap in invertebrates .

What are the proper storage conditions for CYCT1 antibodies?

CYCT1 antibodies should typically be stored at -20°C, where they remain stable for one year after shipment . Many commercial CYCT1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

For optimal preservation of antibody activity:

• Aliquoting is generally unnecessary for -20°C storage

• Some formulations (particularly smaller volumes around 20μl) may contain 0.1% BSA as a stabilizer

• Avoid repeated freeze-thaw cycles which can diminish antibody performance

• Follow manufacturer-specific recommendations, as formulations may vary between suppliers

How is antibody specificity confirmed for CYCT1 and CYCT2?

Antibody specificity for CYCT1 and CYCT2 is typically validated through multiple complementary approaches:

• Western blotting verification: Confirming single band detection at the expected molecular weight (81 kDa for CYCT1)

• Epitope mapping: Many commercial antibodies target specific regions, such as the Cell Signaling Technology antibody produced against a synthetic peptide corresponding to residues surrounding Gly642 of human cyclin T1

• Cross-reactivity testing: Validation against multiple species samples to confirm reactivity with human, mouse, rat, or monkey CYCT1/CYCT2

• siRNA knockdown: Verification by depleting the target protein through siRNA and observing corresponding reduction in antibody signal, as demonstrated in experimental protocols using specific siRNA duplexes for CYCT1 (5′-UUCCGAAUACGUUUCAGCCUGCUUGGA-3′ sense and 5′-AAGGCUUAUGCAAAGUCGGACGAAC-3′ antisense)

How do phosphorylation states affect CYCT1 function and detection?

Phosphorylation plays a critical role in regulating CYCT1 function and assembly into the P-TEFb complex. Research has demonstrated that:

• CYCT1 not bound to CDK9 is rapidly degraded, and productive CYCT1:CDK9 interactions are increased by PKC-mediated phosphorylation of CYCT1

• Specific phosphorylation sites have been identified through mutational analysis. For example, mutation of threonine residues 143 and 149 to alanine (TT143149AA) significantly reduces phosphorylation levels (approximately 4.1-fold decrease) compared to wild-type CYCT1 in the presence of phosphatase inhibitors like okadaic acid

• Phosphorylation state detection can be accomplished through specialized techniques such as in-gel Phospho-Tag staining, which allows visualization of phosphorylated versus non-phosphorylated forms of the protein

• When analyzing CYCT1 by Western blotting, multiple bands or slight molecular weight shifts may indicate different phosphorylation states of the protein rather than non-specific binding

For accurate assessment of phosphorylation states, researchers should consider using phosphatase inhibitors such as okadaic acid during sample preparation and employing phosphorylation-specific detection methods .

What are the methodological considerations for studying CYCT1 promoter activity?

When investigating CYCT1 promoter activity, researchers should consider several methodological approaches based on published protocols:

• Promoter mapping: Studies have identified that the entire promoter activity of the cloned 2,051-nucleotide fragment resides in the 545 nucleotides 5′ to the CYCT1 coding sequence . A promoter containing these 545 nucleotides was at least as active in each tested cell line as was a promoter containing additional 5′ sequences

• Deletion analysis: Sequential deletion analysis has shown that a 205-nucleotide fragment immediately 5′ to the CYCT1 translation initiation site retained 70-100% of the activity of the intact 545-nucleotide promoter

• Cell-type specific effects: Promoter activity varies significantly between cell types. For example, removal of sequences between position −205 and either position −165 or position −120 resulted in fragments that retained 60-100% activity in Jurkat cells but only 20-30% activity in either 293T or U937 cells

• Electroporation protocols: For testing CYCT1 promoter activity in primary cells, electroporation at 320V and 1,500μF has been successfully used with 10μg of luciferase reporter plasmids and 5μg of control plasmid (e.g., pBC12/CMV/lacZ)

• Stimulation conditions: When testing responsiveness to activation, phorbol ester (PMA) can be added at 25ng/ml approximately 2 hours after cell transfection

How can CYCT1 and CYCT2 antibodies be used in chromatin immunoprecipitation studies?

Chromatin immunoprecipitation (ChIP) with CYCT1 antibodies allows researchers to investigate the genomic binding sites of the P-TEFb complex. Based on validated protocols:

• Antibody selection: Use ChIP-validated antibodies like the Cell Signaling Technology Cyclin T1 (D1B6G) Rabbit mAb at recommended dilutions (1:50)

• Protocol optimization: Follow enzymatic ChIP kit protocols (such as SimpleChIP Enzymatic Chromatin IP Kits) that have been validated with the specific antibody

• Sample preparation: For optimal results, use approximately 4 × 10^6 cells per IP reaction

• Advanced techniques: For higher resolution mapping, CUT&RUN and CUT&Tag techniques have been validated for CYCT1 antibodies at 1:50 dilution using specialized assay kits (CUT&RUN Assay Kit #86652 and CUT&Tag Assay Kit #77552)

• Controls: Include appropriate controls such as IgG negative control and antibodies against known transcription factors or chromatin marks as positive controls

These techniques allow researchers to map the genomic locations where CYCT1-containing complexes are actively involved in transcriptional regulation.

What are the key differences in function between CYCT1 and CYCT2 in vivo?

Despite their structural similarities, CYCT1 and CYCT2 display distinct functional roles in vivo:

• Embryonic development: Genetic inactivation of CYCT2 leads to embryonic lethality in mice, demonstrating that CYCT1 cannot compensate for CYCT2 function during development

• HIV transcription: CYCT1 specifically serves as an essential cofactor for HIV Tat protein, promoting RNA Pol II activation and viral gene transcription, while CYCT2 does not effectively support this function

• Gene regulation: Studies indicate that CYCT1 and CYCT2 regulate different subsets of genes important for embryonic development

• Species differences: In C. elegans, individual inactivation of either CYCT1 or CYCT2 had no effect, while simultaneous inactivation of both cyclins produced a severe phenotype equivalent to RNA polymerase II inactivation . This contrasts with mammals, where the cyclins have more distinct functions

• Immunological role: In CYCT1^−/− mice expressing residual wild-type protein, only minor immunological defects were observed, suggesting partial functional contribution to immune cell development

These findings collectively demonstrate that CYCT1 and CYCT2 are not functionally redundant in mammals, with each controlling distinct transcriptional programs.

How does CYCT1 expression vary between resting and activated immune cells?

The regulation of CYCT1 expression in immune cells has important implications for HIV research and understanding transcriptional control during immune cell activation:

• Constitutive expression: Contrary to earlier assumptions, CYCT1 is expressed at significant levels in unstimulated primary lymphocytes, with only slightly lower steady-state expression compared to phorbol ester-treated cells or immortalized cell lines

• Protein levels versus activity: CYCT1 is expressed at sufficient levels in unstimulated primary cells to support robust Tat activity, suggesting that low CYCT1 expression is not the primary limiting factor for HIV gene expression in resting cells

• Transcriptional regulation: CYCT1 promoter activity shows modest enhancement in response to cell activation, indicating that increased CYCT1 function during activation may involve post-translational mechanisms rather than transcriptional upregulation

• Mechanistic implications: These findings challenge the hypothesis that low CYCT1 expression levels in resting cells are responsible for HIV-1 latency, suggesting alternative mechanisms must be involved

What are the optimal approaches for detecting CYCT1 by Western blotting?

For optimal Western blot detection of CYCT1, researchers should follow these evidence-based recommendations:

• Antibody selection: Use validated antibodies at appropriate dilutions (1:2000-1:16000 for Proteintech antibody #20992-1-AP or 1:1000 for Cell Signaling Technology #81464)

• Sample preparation: Prepare cell lysates using appropriate buffer systems. Published protocols have used direct lysis with protein-loading buffer followed by concentration determination using bicinchoninic acid protein assay kit (Pierce)

• Sample processing: Dilute lysates to equal protein concentration, mix with Laemmli sample buffer, and boil for 5 minutes prior to gel loading

• Detection strategy: The expected molecular weight for CYCT1 is 81 kDa, so use appropriate molecular weight markers and optimize exposure time to detect this specific band

• Positive controls: Include lysates from cell lines known to express CYCT1 such as HeLa, A431, Jurkat, K-562, or Y79 cells as positive controls

• Validation approach: For specificity confirmation, consider parallel detection with antibodies targeting other P-TEFb complex components such as CDK9 (such as Santa Cruz Biotechnology sc-484)

How can researchers generate and validate CYCT1/CYCT2 knockdown models?

For studying CYCT1 and CYCT2 function through knockdown approaches:

• siRNA design for CYCT1: Validated siRNA duplexes for CYCT1 include sequences such as 5′-UUCCGAAUACGUUUCAGCCUGCUUGGA-3′ (sense) and 5′-AAGGCUUAUGCAAAGUCGGACGAAC-3′ (antisense)

• CYCT2 knockdown: Published protocols have used siRNA pools from commercial sources (such as Santa Cruz Biotechnology) targeting different regions of mouse CYCT2 mRNA

• Transfection protocols: Typical protocols involve harvesting cells 48 hours post-transfection, with half used for protein analysis and half for RNA extraction

• Control siRNA: Use non-targeting control siRNA containing RNA duplexes that do not match any sequence from the target genome (e.g., mouse genome)

• Validation methods:

  • Protein level: Western blotting with anti-CYCT1, anti-CYCT2, and anti-CDK9 antibodies

  • mRNA level: RNA extraction with Trizol followed by RNeasy column purification and quantitative RT-PCR

• Gene trap approach: For stable genetic models, gene trap insertion has been successfully used to create CYCT2-deficient mice. For instance, insertion in intron 7 created a fusion protein between the N-terminus of CYCT2 (235 residues) and β-Geo (1,323 residues)

What are the recommended antigen retrieval methods for CYCT1 immunohistochemistry?

For optimal CYCT1 detection in tissue sections by immunohistochemistry:

• Buffer selection: TE buffer at pH 9.0 is suggested as the primary antigen retrieval method for CYCT1 antibodies

• Alternative approach: Citrate buffer at pH 6.0 can be used as an alternative antigen retrieval method if TE buffer does not yield optimal results

• Dilution optimization: Use antibody dilutions in the range of 1:50 to 1:500, with specific optimization required for each tissue type and fixation method

• Positive control tissues: Human ovary cancer tissue has been validated for CYCT1 antibody testing in IHC applications

• Detection systems: Choose chromogenic or fluorescent detection systems based on research needs, with appropriate secondary antibodies matched to the host species of the primary antibody

• Titration recommendation: It is advised that CYCT1 antibodies be titrated in each testing system to obtain optimal results, as sensitivity can be sample-dependent

How can CYCT1 phosphorylation states be effectively analyzed?

For researchers investigating CYCT1 phosphorylation:

• Phosphatase inhibition: Include okadaic acid (1μM) during sample preparation to preserve phosphorylation states

• Detection methods: In-gel Phospho-Tag staining provides specific visualization of phosphorylated versus non-phosphorylated forms of CYCT1

• Mutation analysis: Site-directed mutagenesis of key phosphorylation sites (such as threonine residues 143 and 149) to alanine can help determine the functional significance of specific phosphorylation events

• Expression systems: Both full-length and truncated versions of CYCT1 have been successfully used to study phosphorylation, with CycT1(192) and CycT1(280) constructs showing similar phosphorylation patterns

• Controls: Include unphosphorylated proteins such as BSA as negative controls for phosphorylation-specific detection methods

How are CYCT1 antibodies utilized in HIV-1 research?

CYCT1 antibodies serve several critical functions in HIV-1 research:

• Tat interaction studies: As CYCT1 is an essential cofactor for HIV Tat protein, antibodies help investigate the binding between CYCT1 and Tat, which promotes RNA Pol II activation and viral gene transcription

• Latency mechanisms: Research with CYCT1 antibodies has challenged previous assumptions that low CYCT1 expression restricts HIV-1 expression and replication in resting CD4+ T cells, showing that CYCT1 is expressed at sufficient levels in unstimulated cells to support Tat activity

• P-TEFb complex analysis: Antibodies allow detection of CYCT1 in the context of the P-TEFb complex, helping to understand how HIV hijacks cellular transcription machinery

• Transcriptional regulation: ChIP experiments using CYCT1 antibodies help map genomic locations where CYCT1-containing complexes regulate HIV proviral transcription

• Therapeutic target assessment: CYCT1 antibodies support research into potential therapeutic approaches targeting the Tat-CYCT1 interaction to inhibit HIV transcription

What is the significance of CYCT1 and CYCT2 in developmental biology research?

The distinct roles of CYCT1 and CYCT2 in development have been elucidated through genetic studies:

• Embryonic lethality: CYCT2 knockout mice exhibit embryonic lethality, demonstrating its essential role in mammalian development that cannot be compensated by CYCT1

• Functional non-redundancy: Despite structural similarities, CYCT1 and CYCT2 regulate different subsets of genes important for embryonic development

• Species differences: In C. elegans, individual inactivation of either CYCT1 or CYCT2 had no effect, while simultaneous inactivation produced a phenotype equivalent to RNA polymerase II inactivation, revealing evolutionary differences in cyclin function

• Developmental transcription: CYCT1 and CYCT2, as components of distinct P-TEFb complexes, regulate specific transcriptional programs during development through CDK9-mediated phosphorylation of RNA polymerase II

• Tissue-specific functions: Differing expression patterns and functions of CYCT1 and CYCT2 across tissues suggest specialized roles in tissue development and homeostasis

These findings emphasize the importance of studying both cyclins to understand developmental transcriptional regulation.

What methodological approaches are used to study CYCT1 promoter regulation?

Research into CYCT1 promoter regulation has utilized several specialized techniques:

• Promoter cloning: The GenomeWalker Kit has been used to clone the promoter region of CYCT1, with nested PCR amplification using gene-specific primers (such as T1GP1: 5′-TTTGTTGTTGTTCTTCCTCTCTCCCTC-3′ and T1GP2: 5′-CTCCATAGTGCTTCAACCAGAAGGCAG-3′)

• Transcription factor binding prediction: Computational tools such as MacVector 6.5.1 and Transcription Element Search Software (TESS) have been employed to identify putative transcription factor binding sites in the CYCT1 promoter

• Deletion analysis: Sequential deletion constructs have been used to map critical regulatory regions within the promoter, revealing that a 205-nucleotide fragment immediately 5′ to the translation initiation site retained 70-100% of full promoter activity

• Cell-type comparison: Testing promoter activity across multiple cell lines (Jurkat, 293T, U937) has revealed cell-type specific regulation patterns, with differential effects of promoter deletions based on cell type

• Reporter assays: Luciferase reporter plasmids containing CYCT1 promoter fragments have been used to quantitatively assess promoter activity, with co-transfection of β-galactosidase expression plasmids as internal controls

These methodologies provide a framework for investigating transcriptional regulation of CYCT1 in various cellular contexts.

How do CYCT1 and CYCT2 function in the context of P-TEFb complex assembly?

The assembly and function of P-TEFb complexes containing CYCT1 or CYCT2 involve several regulatory mechanisms:

• Protein stability regulation: CYCT1 not bound to CDK9 is rapidly degraded, indicating that complex formation stabilizes the cyclin component

• Phosphorylation-mediated assembly: PKC-mediated phosphorylation of CYCT1 increases productive CycT1:CDK9 interactions, representing a regulatory mechanism for P-TEFb complex assembly

• Specific phosphorylation sites: Threonine residues 143 and 149 on CYCT1 are important phosphorylation sites that affect complex formation, as demonstrated by reduced phosphorylation in TT143149AA mutants

• Functional differences: Despite similar roles in P-TEFb complex formation, CYCT1 and CYCT2 direct the complex to different gene targets, as evidenced by their non-redundant functions in development

• Gene-specific regulation: The distinct P-TEFb complexes containing either CYCT1 or CYCT2 regulate different subsets of genes important for cellular processes and development

Understanding these mechanisms provides insight into how cells regulate transcriptional elongation through differential assembly of P-TEFb complexes.

What are the best practices for immunofluorescence detection of CYCT1?

For optimal immunofluorescence detection of CYCT1:

• Cell types: MCF-7 cells have been validated for immunofluorescence applications with CYCT1 antibodies

• Antibody dilution: Use dilutions in the range of 1:50 to 1:500, with specific optimization required for each cell type and fixation protocol

• Nuclear localization: Expect nuclear localization of CYCT1 signal, consistent with its function in transcriptional regulation

• Fixation methods: Standard fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100, though specific protocols may vary based on antibody requirements

• Controls: Include negative controls (secondary antibody only) and positive controls (cells known to express high levels of CYCT1)

• Co-localization studies: Consider co-staining with other P-TEFb components such as CDK9 to demonstrate complex formation or with markers of transcriptional activity