Phospho-TBC1D4 (T642) Antibody
Product Specs
Target Background
- The C-terminal region (CTR) is largely alpha-helical and mediates TBC1D4 RabGAP dimerization. PMID: 28545963
- Disruption of TBC1D4 is prevalent among North American Inuit, resulting in exclusively elevated postprandial glucose. This leads to underdiagnosis of type 2 diabetes, unless an oral glucose tolerance test (OGTT) is performed. PMID: 27561922
- AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle. PMID: 27152871
- AS160 and TBC1D1 phosphorylations were evident 30 minutes after exercise. PMID: 24876356
- Individuals homozygous for a nonsense p.Arg684Ter variant exhibit markedly higher concentrations of plasma glucose and serum insulin 2 hours after an oral glucose load compared to individuals with other genotypes. PMID: 25043022
- Findings indicate that a reduction in insulin-induced phosphorylation of AS160 on specific sites in skeletal muscle contributes to the insulin resistance observed in a sedentary aging population. PMID: 23801578
- Insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160. PMID: 22028408
- AS160 interacts with the large cytoplasmic NP domain of the alpha-subunit of the Na(+),K(+)-ATPase. AMP-stimulated protein kinase (AMPK) and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase. PMID: 20943949
- Crystal structures of human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating protein (RabGAP) domains reveal critical elements for GLUT4 translocation. PMID: 21454505
- AS160 phosphotyrosine-binding domain constructs inhibit insulin-stimulated GLUT4 vesicle fusion with the plasma membrane. PMID: 21454690
- Impaired insulin-induced site-specific TBC1D4 phosphorylation may contribute to skeletal muscle insulin resistance in type 2 diabetes. PMID: 20938636
- Results demonstrate that AS160 phosphorylation level is frequently elevated in breast cancer; these findings suggest a potential role of AS160 in breast tumorigenesis and indicate that p-AS160 might serve as a marker and a potential novel therapeutic target. PMID: 20574165
- WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complex. PMID: 20937822
- S711 is a novel TBC1D4 phosphorylation site regulated by AMPK in skeletal muscle. PMID: 19923418
- KIAA0603 is likely to be a Rab GAP that participates in the regulation of activated T cells in atopic dermatitis. PMID: 15304337
- This study investigated the expression of and in vivo insulin action on AS160 in skeletal muscle of normal and type 2 diabetic patients. PMID: 15919790
- Results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation. PMID: 15971998
- AS160 is phosphorylated in a time-dependent manner during moderate-intensity exercise. PMID: 17077344
- Regulation of AS160 and interaction with 14-3-3 in skeletal muscle are influenced by resistance exercise and insulin but do not fully explain the effect of resistance exercise on whole-body insulin action. PMID: 17369524
- Effects of endurance exercise training on insulin signaling and AS160 in human skeletal muscle. PMID: 17513702
- AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases. PMID: 17617058
- Impaired insulin signaling through Akt and AS160 in part explains insulin resistance at the molecular level in skeletal muscle in polycystic ovary syndrome. PMID: 17977950
- AS160, previously recognized as a key player in insulin signaling in skeletal muscle and adipose tissue, is also a major effector of protein kinase B/Akt signaling in the beta-cell. PMID: 18276765
- The transcript variant 2 of AS160, in contrast to full-length AS160, is a novel regulator of glucose transport that positively influences glucose-uptake rates. PMID: 18771725
- Muscle TBC1D4 phosphorylation across the leg is increased during recovery following resistance exercise. PMID: 18845784
- Specific phosphorylation of TBC1D4 in human skeletal muscle in response to physiological exercise-induced hyperinsulinemia. PMID: 19252894
- Prematurely truncated TBC1D4 protein tended to increase basal cell membrane GLUT4 levels (P = 0.053) and significantly reduced insulin-stimulated GLUT4 cell membrane translocation (P < 0.05). PMID: 19470471
Customer Reviews
Applications : /
Sample type: cells
Review: P-AS160 was purchased from CUSABIO. (PA080083, CUSABIO, 1:1000).
Q&A
Basic Research Questions
How do I validate antibody specificity for TBC1D4-T642 phosphorylation in muscle tissue samples?
Validation requires a multi-step approach:
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Use knockout controls (e.g., TBC1D4-deficient myocytes) to confirm absence of signal
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Perform peptide competition assays with phosphorylated vs. non-phosphorylated T642 peptides
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Combine with phosphatase treatment of lysates to demonstrate signal loss
Critical data interpretation table:
What experimental conditions optimize T642 phosphorylation detection in insulin signaling studies?
Optimal protocol parameters:
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Stimulation: 100 nM insulin for 15-30 min (maximal Akt activation)
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Lysis buffer: Include phosphatase inhibitors (1 mM Na3VO4, 10 mM β-glycerophosphate) and protease inhibitors
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Gel percentage: 8% SDS-PAGE for clear separation from TBC1D1 (160 vs 150 kDa)
Sample preparation note: Skeletal muscle requires mechanical homogenization with ceramic beads in RIPA buffer containing 1% SDS to disrupt myofibrillar structures .
Advanced Research Challenges
How to resolve contradictory findings between T642 phosphorylation and GLUT4 translocation in exercise models?
Key methodological considerations:
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Temporal resolution: Collect serial biopsies (pre, 0h, 1h, 2h post-exercise) as phosphorylation peaks at 2h while GLUT4 recycling occurs earlier
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Compartmentalization analysis: Use membrane fractionation + immunofluorescence to detect spatial phosphorylation patterns
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Kinase crosstalk: Employ AMPKα2-KD mice to isolate Akt vs. AMPK effects on T642
Example conflict resolution:
Contraction-induced GLUT4 translocation without T642 phosphorylation may involve:
What strategies distinguish TBC1D4-T642 phosphorylation from homologous sites in TBC1D1?
How to interpret discrepant phosphorylation kinetics between in vitro and in vivo models?
Critical factors causing divergence:
Resolution protocol:
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Perform dose-response curves with physiological (1-10 nM) vs. supraphysiological (100 nM) insulin
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Use Akt2-specific inhibitors (e.g., MK-2206) to isolate pathway-specific effects
Technical Optimization
What validation controls are essential when using Phospho-T642 antibodies in cancer metabolism studies?
Essential controls for tumor models:
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Isoform-specific siRNA: Concurrent knockdown of TBC1D4 vs. TBC1D1
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Orthotopic xenografts: Compare phosphorylation in tumor vs. adjacent normal tissue
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Hypoxia mimetics: Test CoCl2 (200 μM) effects on pseudo-phosphorylation
Critical data interpretation caveats:
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Many carcinomas overexpress insulin receptor → artifactual phosphorylation in serum-starved conditions
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Stromal cell contamination (>15%) obscures tumor-specific signals
How to design longitudinal studies examining T642 phosphorylation dynamics in metabolic syndrome models?
Optimal sampling framework:
| Timepoint | Metabolic Challenge | Tissue Collection |
|---|---|---|
| Baseline | Overnight fast | Muscle biopsy (vastus lateralis) |
| 15 min | Hyperinsulinemic-euglycemic clamp | Serial biopsies (contralateral leg) |
| 4 hr | Mixed-meal challenge | Plasma + tissue analysis |
Key analytical tools:
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Multiplexed imaging: CODEX® platform for spatial phosphorylation analysis
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Phosphoproteomics: TiO2 enrichment + LC-MS/MS with DIA acquisition
Mechanistic Investigations
What experimental approaches link T642 phosphorylation status to Rab GTPase activity?
Functional assays:
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GAP activity measurement:
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Subcellular trafficking:
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Structural biology:
How to investigate compensatory phosphorylation networks when T642 is genetically ablated?
Systems-level analysis workflow:
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Phosphoproteomics: TMT-labeled MS with TiO2 enrichment (≥10,000 phosphosites)
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Kinase activity profiling: PamChip® peptide arrays + 384-kinase inhibitor panel
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Molecular dynamics: 500ns simulations of TBC1D4 mutant structures
Key findings from T642A knockin models:
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AMPK-dependent S711 phosphorylation becomes dominant GLUT4 regulator
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RAB10 GAP activity maintained through 14-3-3 binding at S318
Translational Considerations
What human validation strategies confirm murine findings on T642 phosphorylation in diabetes pathology?
Multi-ethnic validation framework:
Critical technical adaptation:
