TNK1 Antibody

Basic Research Questions

• What is TNK1 and why is it significant in research?

  TNK1 (Tyrosine Kinase Non-Receptor 1) is a 72.5 kDa non-receptor protein tyrosine kinase that plays crucial roles in cellular signaling pathways. It contains a sterile alpha motif (SAM), a tyrosine kinase catalytic domain, an SH3 domain, and a unique C-terminal region containing a ubiquitin association domain (UBA) . TNK1 is significant in research due to its dual nature - functioning as both a tumor suppressor by negatively regulating cell growth through Ras inhibition and potentially as an oncogenic factor in certain contexts . TNK1 has been implicated in various pathways including IFN signaling, antiviral immunity, and inflammatory responses , making it a compelling target for studies in cancer biology, immunology, and inflammatory diseases.

• What are the most reliable applications for TNK1 antibodies in laboratory research?

  Based on validated research, TNK1 antibodies are most reliably used in:

  Application	Validation Level	Notes
  Western Blot (WB)	Highly validated	Optimal at dilutions of 1:1000-1:6000
  ELISA	Well-validated	Commonly used for quantification
  Immunohistochemistry (IHC)	Validated	Effective at dilutions of 1:50-1:500
  Immunoprecipitation (IP)	Validated	Useful for studying protein interactions
  Immunofluorescence (IF)	Validated	Helpful for cellular localization studies

  When selecting antibodies, researchers should prioritize those with validation in their specific application and species of interest. Cell Signaling Technology's TNK1 (C44F9) Rabbit mAb has been cited in multiple publications for Western blot applications , suggesting strong reliability.

• How is TNK1 regulated at the transcriptional and post-translational levels?

  Transcriptional regulation:

  • The TNK1 promoter lacks conventional TATA, CAAT, or initiator elements but contains multiple transcription start sites

  • Transcription is initiated by a TATA-like element composed of an AT-rich sequence at -30bp from the major transcription start site

  • Key transcription factors include Sp1, Sp3, AP2, and MED1, which bind to three GC boxes in the proximal promoter

  • Cellular stress (like serum withdrawal) increases high-affinity interactions between these factors and the TNK1 promoter, leading to increased expression

  Post-translational regulation:

  • Phosphorylation: MARK-mediated phosphorylation at S502 promotes interaction with 14-3-3 proteins, which sequesters TNK1 and inhibits its kinase activity

  • Ubiquitin interaction: TNK1 contains a C-terminal ubiquitin-association domain (UBA) that binds to polyubiquitin with high affinity; this interaction is crucial for TNK1 activation

  • Regulation by cellular localization: TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states

  • TNK1 clustering in ubiquitin-rich puncta correlates with its activation

• What expression patterns of TNK1 are observed across different tissues and disease states?

  Normal tissue expression:

  • High expression in fetal tissues including lung, liver, brain, and kidney

  • Ubiquitous but variable expression in adult tissues

  • The alternate spliced variant of TNK1/Kos1 (47 kDa) is ubiquitously expressed in undifferentiated murine ES cells, mouse embryos, and adult tissues

  Disease-associated expression patterns:

  • Downregulation in diffuse large B-cell lymphoma (DLBCL) patients compared to normal B-lymphocytes

  • Allelic loss and/or downregulation in approximately 22% of newly diagnosed DLBCL patients

  • Upregulation in atherosclerotic inflammation, including both high-fat diet-fed ApoE(-/-) mice aorta and human ruptured plaques

  • Expression of a 60 kDa truncated fusion TNK1-C17orf61 gene product in the L540 Hodgkin Lymphoma cell line

  • TNK1 dependencies identified in a subset of primary hematological cancers including acute myeloid leukemia (AML), B-cell and T-cell acute lymphoblastic leukemia (ALL), and chronic myelogenous leukemia (CML)

• What positive and negative controls should be used when working with TNK1 antibodies?

  Positive controls:

  • Cell lines: K-562 cells, HL-60 cells, and PC-3 cells have been validated for Western blot detection of TNK1

  • Tissues: Human prostate cancer tissue and human liver tissue have been validated for IHC detection

  • Recombinant TNK1: Purified protein from Sf9 insect cells expressing TNK1 ΔCT (1-510aa) with N-terminal 6x-His and GST tags

  Negative controls:

  • TNK1 knockout or knockdown models: TNK1/Kos1 knockout mice tissues

  • shRNA-mediated TNK1 knockdown in THP-1 cells

  • Samples treated with non-targeting antibodies of the same isotype

  • Secondary antibody-only controls to assess non-specific binding

  Additional validation approaches:

  • Peptide competition assays using the immunogen peptide

  • Antibody validation in multiple applications (WB, IHC, IF) to ensure consistent results

  • Cross-validation with multiple antibodies targeting different epitopes of TNK1

Advanced Research Questions

• How can researchers effectively study the dual tumor suppressor/oncogenic functions of TNK1?

  TNK1 exhibits context-dependent tumor suppressor or oncogenic functions. To effectively study this duality:

  For tumor suppressor functions:

  • Use TNK1/Kos1 knockout mouse models, which develop spontaneous tumors (B-cell lymphomas, DLBCL, and hepatocellular carcinomas)

  • Analyze Ras activation status in TNK1-deficient tissues, as TNK1 negatively regulates Ras by destabilizing the RasGEF (Grb2-Sos1) complex

  • Investigate allelic loss of TNK1 in human cancer samples using SNP arrays and gene expression profiling

  • Examine TNK1 promoter methylation as a potential mechanism for TNK1 downregulation

  For oncogenic functions:

  • Study the L540 Hodgkin lymphoma cell line, which expresses a 60 kDa truncated fusion TNK1-C17orf61 gene product

  • Investigate TNK1 dependencies in primary cancer samples using RNAi screening approaches

  • Examine the effects of releasing TNK1 from 14-3-3 inhibition, which enhances TNK1-driven proliferation in a UBA-dependent manner

  • Test selective TNK1 inhibitors (like TP-5801) on TNK1-dependent cancer cells in vitro and in vivo

  Experimental approach integration:

  • Compare wild-type TNK1 with kinase-dead mutants to determine the role of kinase activity in different contexts

  • Analyze the phosphorylation status of TNK1 using phospho-specific antibodies

  • Investigate the interactions between TNK1 and key signaling proteins in both tumor suppression (Ras pathway) and oncogenesis

• What are the optimal experimental conditions for detecting TNK1 phosphorylation states?

  Detecting TNK1 phosphorylation requires careful consideration of experimental conditions:

  Sample preparation:

  • Rapidly harvest cells/tissues and immediately lyse in phosphatase inhibitor-containing buffer to preserve phosphorylation status

  • For tissues, use snap-freezing in liquid nitrogen followed by homogenization in cold lysis buffer

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) in all buffers

  Phospho-specific antibody selection:

  • Use phospho-specific antibodies that target key regulatory phosphorylation sites, particularly S502 which mediates 14-3-3 binding

  • Consider phospho-tyrosine antibodies to detect TNK1 auto-phosphorylation, which correlates with kinase activity

  Detection methods:

  • Western blot: Use 7.5% or 10% SDS-PAGE gels for optimal separation of phosphorylated forms

  • Immunoprecipitation followed by phospho-specific Western blot for enhanced sensitivity

  • Phos-tag™ SDS-PAGE to enhance the mobility shift of phosphorylated proteins

  Controls and validation:

  • Treatment with phosphatase (e.g., lambda phosphatase) as a negative control

  • Use of cellular stress conditions (serum starvation) which are known to affect TNK1 phosphorylation

  • Comparison with kinase-dead TNK1 mutants to distinguish between auto-phosphorylation and phosphorylation by other kinases

• How can researchers effectively investigate the interaction between TNK1 and its binding partners?

  To study TNK1 interactions with partners like 14-3-3 proteins, ubiquitin, and other signaling molecules:

  Co-immunoprecipitation approaches:

  • Use TNK1 antibodies for immunoprecipitation followed by Western blot for interacting proteins

  • Reverse co-IP with antibodies against suspected binding partners (14-3-3, Grb2)

  • For studying 14-3-3 interactions, compare wild-type TNK1 with S502 mutants that cannot be phosphorylated

  Advanced interaction assessment technologies:

  • BioID proximity labeling (BirA fused to TNK1) followed by mass spectrometry to identify interacting proteins in cellular context

  • Use of phospho-binding defective 14-3-3 (K49Q) mutants as negative controls for phosphorylation-dependent interactions

  • FRET or BiFC assays to visualize interactions in living cells

  For ubiquitin binding studies:

  • Use purified TNK1 UBA domain in pull-down assays with different ubiquitin chain types

  • Test TNK1 variants with point mutations that disrupt ubiquitin binding

  • Analyze TNK1 localization to ubiquitin-rich puncta using immunofluorescence and co-localization studies

  Functional validation:

  • Correlate binding interactions with TNK1 kinase activity measurements

  • Assess the impact of disrupting specific interactions on downstream signaling events

  • Evaluate cellular phenotypes (proliferation, survival) when interactions are enhanced or disrupted

• What are the methodological considerations for studying TNK1's role in immune signaling pathways?

  TNK1 has been implicated in IFN signaling and antiviral immunity . Key methodological considerations include:

  Cell systems for immune studies:

  • THP-1 cells show high TNK1 expression compared to other atherosclerotic-related cells (HUVEC, HBMEC, HA-VSMC)

  • Primary immune cells (particularly B cells) are relevant based on the B-cell lymphoma phenotype in TNK1 knockout mice

  • Hepatocytes are important for studying TNK1's role in IFN signaling and antiviral immunity

  Pathway analysis approaches:

  • Monitor JAK-STAT signaling: Assess STAT1 phosphorylation and activation in response to IFNs with and without TNK1

  • Evaluate IFN-stimulated gene (ISG) expression using qRT-PCR or RNA-seq following TNK1 modulation

  • Analyze NF-κB pathway activation and pro-inflammatory cytokine production (IL-12, IL-6, TNF-α)

  Functional immune assays:

  • Viral infection models with TNK1 knockdown/knockout to assess antiviral defense capabilities

  • Lipid uptake and cholesterol content assays in macrophages with modulated TNK1 expression

  • Cytokine production assays following stimulation (e.g., with oxLDL in macrophages)

  In vivo models:

  • TNK1 knockout mice challenged with viral infections or inflammatory stimuli

  • ApoE(-/-) mice fed high-fat diet as a model for atherosclerotic inflammation and TNK1 upregulation

  • Evaluation of human carotid endarterectomy atherosclerotic plaques for TNK1 expression in stable versus ruptured states

• How can TNK1 kinase activity be accurately measured in research settings?

  Several approaches can be used to measure TNK1 kinase activity:

  In vitro kinase assays:

  • Use purified recombinant TNK1 ΔCT (1-510aa) expressed in Sf9 insect cells

  • WASP peptide has been identified as an efficient peptide substrate for TNK1

  • Monitor phosphorylation using radiometric (³²P-ATP) or non-radiometric (ELISA-based) detection methods

  Inhibitor profiling:

  • ATP-competitive inhibitors can be tested against TNK1, with the Ack1 inhibitor (R)-9b showing complete inhibition at 10 μM (IC₅₀ of 470 nM)

  • EGFR family inhibitors (erlotinib, gefitinib) show 30-40% inhibition at 10 μM

  • TP-5801 is a selective TNK1 inhibitor with nanomolar potency

  Cellular activity measurements:

  • Immunoprecipitate TNK1 from cells followed by in vitro kinase assays

  • Monitor auto-phosphorylation status as a proxy for activity

  • Evaluate phosphorylation of downstream targets in cellular context

  Controls and validation:

  • Compare wild-type TNK1 with kinase-dead mutants

  • Use known activating conditions (release from 14-3-3 binding) or inhibitory conditions (MARK-mediated phosphorylation at S502)

  • Correlate kinase activity with cellular phenotypes (e.g., growth factor-independent proliferation)

• What are the best approaches for studying TNK1's subcellular localization and translocation?

  TNK1's subcellular localization is critical to its function, particularly its translocation between 14-3-3-bound (inactive) and ubiquitin-rich puncta (active) states :

  Immunofluorescence microscopy techniques:

  • Use TNK1 antibodies validated for immunofluorescence (IF) applications

  • Co-staining with markers for ubiquitin-rich puncta, 14-3-3 proteins, and cellular compartments

  • Live-cell imaging using fluorescently tagged TNK1 to monitor dynamic changes in localization

  Biochemical fractionation:

  • Separate nuclear, cytoplasmic, membrane, and cytoskeletal fractions

  • Western blot analysis of TNK1 distribution across fractions

  • Compare distribution under different conditions (e.g., serum starvation, IFN stimulation)

  Manipulating TNK1 localization:

  • S502A mutation to prevent 14-3-3 binding and enhance ubiquitin-rich puncta localization

  • UBA domain mutations to disrupt ubiquitin binding

  • Pharmacological manipulation of MARK kinases to modulate S502 phosphorylation

  Advanced imaging approaches:

  • Super-resolution microscopy for detailed analysis of TNK1 clustering in ubiquitin-rich puncta

  • FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics of TNK1 association with different cellular compartments

  • Correlative light and electron microscopy to identify the precise subcellular structures where TNK1 localizes