Phospho-ERBB2 (T686) Antibody
Description
Target and Biological Context
Phospho-ERBB2 (T686) Antibody specifically recognizes HER2 phosphorylated at threonine 686 (T686), a residue within the receptor's juxtamembrane domain. HER2 is a tyrosine kinase receptor overexpressed in cancers like gastric and breast cancer, driving cell proliferation and survival through dimerization with other HER family members (e.g., EGFR) and downstream signaling activation . Phosphorylation at T686 by kinases such as type II cGMP-dependent protein kinase (PKG II) modulates HER2 activity, often exerting inhibitory effects .
Mechanism of T686 Phosphorylation
PKG II directly binds HER2 and phosphorylates T686, which suppresses ligand-induced tyrosine phosphorylation (activation) of HER2. This contrasts with other kinases like protein kinase C (PKC) or protein kinase A (PKA), which phosphorylate T686 but enhance HER2 signaling . Key experimental findings include:
Functional Implications in Cancer
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Inhibition of HER2 Activation: T686 phosphorylation by PKG II disrupts HER2 dimerization and downstream signaling (e.g., MAPK, PI3K/AKT), reducing tumor cell proliferation .
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Therapeutic Potential: Dual inhibition of HER2 and EGFR via PKG II-mediated phosphorylation offers a promising strategy for cancers resistant to single-target therapies .
Research Applications
This antibody is utilized in:
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Western Blotting: Detects endogenous T686-phosphorylated HER2 in cell lines (e.g., A431 carcinoma cells) under EGF stimulation .
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Functional Studies: Evaluates HER2 activation status in PKG II-overexpressing systems or gastric cancer models .
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Mechanistic Insights: Distinguishes PKG II-specific phosphorylation effects from other kinases (PKC/PKA) .
Comparative Analysis of T686 Phosphorylation
| Kinase | Effect on HER2 | Biological Outcome |
|---|---|---|
| PKG II | Inhibits tyrosine phosphorylation | Reduces tumor growth and metastasis |
| PKC/PKA | Enhances activation/internalization | Promotes cell proliferation |
Product Specs
Target Background
- Anionic porphyrin's abnormally sensitive electrochemical sensing performance for DNA sequences specific to the HER2 gene holds significant promise for tumor diagnosis and treatment. PMID: 30340409
- The study demonstrated that mRNA and protein levels of COX2 and HER2 were upregulated in CRC compared to the adjacent tissues. COX2 protein levels and nuclear COX2 expression were correlated with poor prognosis in CRC patients. COX2 expression was positively associated with HER2 expression. PMID: 29873317
- In patients with HER2-positive advanced breast cancer who have undergone extensive pretreatment with anti-HER2 agents and cytotoxic chemotherapy, trastuzumab emtansine (T-DM1) demonstrated good tolerability and provided a significant progression-free survival of 6 months and an overall survival that has not yet been reached. PMID: 29326401
- The expression of C-Met and HER2 protein in lung adenocarcinoma is highly correlated. Further research is warranted to investigate whether their combined targeting could offer synergistic benefits in the treatment of lung adenocarcinoma. PMID: 29400000
- Although ST6GalI overexpression led to increased HER2 sialylation, resulting in decreased HER2 phosphorylation, high alpha2,6-sialylation enhanced Akt and ERK phosphorylation levels compared to those in the vector cell line. Conversely, ST6GalI knockdown exhibited opposite effects. Collectively, these findings suggest a functional role of ST6GalI in promoting tumor cell progression and trastuzumab resistance. PMID: 30226606
- This study demonstrates that miR-495 exerts promotive effects on GC chemosensitivity by inactivating the mTOR signaling pathway through suppression of ERBB2. The study provides robust evidence supporting the potential use of miR-495 as a novel therapeutic target in the chemotherapy of GC. PMID: 30147110
- In early breast cancer, PIK3CA mutations appear to identify HER2+ patients who are less likely to achieve pCR. The clinical implications of PIK3CA mutations tend to vary between exon 9 and exon 20. This mechanism warrants further investigation in subsequent studies. PMID: 29575819
- HER2 and HER3 expression were detected in 22.2% and 86.1% of samples, respectively. The frequency of EGFR mutation was 45.7% and did not significantly differ between stage 0 and IA1 (40.0% and 48.0%, respectively), suggesting that EGFR mutation is not correlated with cancer progression from stage 0 to IA1. PMID: 29473311
- Studies have shown that the heterogeneity of HER2 expression accelerates the development of metastases, leading to poor survival in mice with heterogeneous HER2 expression (HER2-60). PMID: 30042341
- Her-2/neu amplification increases with the increasing grade of breast cancer. A high proportion of Her-2/neu gene amplified cases indicates aggressive disease in that area and necessitates FISH testing on a large scale, which is the gold standard for equivocal cases on immunohistochemistry. PMID: 30060783
- Data indicate that the primary mechanism involves the ability of p140Cap to interfere with ERBB2-dependent activation of Rac GTPase-controlled pathways. PMID: 28300085
- The study showed that the expression levels of Gli1 and HER2 were significantly higher in gastric cancer and are positively related. HER2 may regulate Gli1 through the Akt-mTOR-p70S6K pathway. PMID: 29321573
- The combination of immunohistochemical expression of BRCA1, ER, PR, and HER-2/neu along with clinicopathological details may be helpful in identifying individuals more likely to carry BRCA1 mutations, thus selecting candidate individuals and family members for genetic screening for BRCA1 mutations. PMID: 29567881
- In the current settings, HER2/neu is not considered a prognostic marker in head-and-neck cancers. PMID: 30004046
- These results suggest that HE4 expression increases in patients with HER2/neu amplification. PMID: 30004048
- HER2 gene amplification in circulating tumor DNA predicts resistance to trastuzumab emtansine in HER2-positive breast neoplasms. PMID: 29700710
- Statistical analysis performed in this study did not reveal a significant relationship between HER2 overexpression on tumor cells and microvessel density in the tumor stroma. PMID: 30334990
- Data showed a high rate of discordance in matched pairs of primary tumors and metastases, suggesting that accurate evaluation of proto-oncogene protein HER-2 (HER2) status is essential before any therapeutic decision. PMID: 30203148
- HER2 gene amplification occurred during the early stages of gastric cancer and displayed heterogeneity in several cases. HER2 gene amplification may contribute to tumor progression in early gastric cancer. PMID: 30120594
- Activating HER2 mutations are present in approximately 3% of bone metastases from breast cancers, with significantly higher rates in the pleomorphic subtype of lobular cancer. PMID: 30094493
- The results suggest a potential link between tRNALeu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling, particularly in breast cancer. PMID: 28816616
- High HER2 expression is associated with metastasis in breast cancer. PMID: 29187405
- This study confirmed that biosimilar trastuzumab improves the overall response rate when combined with chemotherapy for HER2+ breast cancer. PMID: 30082554
- The authors highlight a gender difference in the prognostic value of concomitant AIB1 and HER2 copy number gain (CNG) in glioma patients, which was previously underappreciated. These observations suggest that genetic alterations, in synergy with essential aspects of sex determination, influence glioma biology and patient outcomes. PMID: 30153912
- The survival rates in this study align with documented global rates. Nodal disease burden emerged as the most significant prognostic factor. Additionally, in EBCs, a lack of hormone receptor expression and in LABC, Her2neu overexpression appear to worsen the outcome. PMID: 30147088
- Results indicate that HER2 and FGFR2 are regulated by DDX6 at the post-transcriptional level in gastric cancer. PMID: 29987267
- HER2 overexpression is associated with Gastric Cancer. PMID: 29938472
- The ERBB2 oncogene at 17q12 is susceptible to palindromic gene amplification in HER2-positive breast tumors. PMID: 28211519
- Results show that mutations in ERBB2-exon17 were associated with worse survival outcomes in patients with pancreatic neoplasm. [review] PMID: 30227250
- High HER2 expression and Gene Amplification are associated with Upper Tract Urothelial Carcinomas. PMID: 28755093
- High HER2 expression is associated with invasion and lymph node metastasis in gastric cancer. PMID: 29970682
- The basal HER2 phenotype exhibited poor DFS but an equivalent pCR rate after concurrent neo-adjuvant chemotherapy with trastuzumab. A distinct treatment approach for the basal-HER2 type is necessary, even for cases that achieved adequate clinical response following neo-adjuvant chemotherapy. PMID: 29971625
- In the largest series reported to date, patients with HER2-amplified m17 cancers treated with trastuzumab have outcomes comparable to patients from the large phase III adjuvant trastuzumab trials who were HER2-positive. This supports the critical role of HER2-directed therapy in this patient population. PMID: 28986743
- The interplay of dual MET/HER2 overexpression in the AKT and ERK pathways for esophageal cancer is described. Therefore, combination therapy could be a promising strategy for EAC with amplification of both MET and HER2. PMID: 29223420
- This study provides evidence that the hostile environment developed in spheroids plays a pivotal role in the acquisition of resistance to Trastuzumab and is associated with an increase in the number of breast cancer stem cells as well as a modulation in HER2 expression. PMID: 28722778
- A major finding of this study is that one out of five (20%) patients with breast cancer BM exhibited a receptor discrepancy between the primary tumor and subsequent BM. Loss of hormone receptors (ER and/or PR) expression and gain of HER2 overexpression were the most commonly observed changes. PMID: 28975433
- High HER2 expression is associated with Gastric Adenocarcinoma. PMID: 29802704
- Absence of HER2 Expression of Circulating Tumor Cells is associated with Non-Metastatic Esophageal Cancer. PMID: 30275185
- HER2 positivity was observed in a minority of rectal cancer patients and was not significantly associated with clinicopathologic and molecular characteristics. PMID: 30056472
- This study discovered a novel enhancer, the HER2 gene body enhancer (HGE), located in the 3' gene body of HER2. The HGE activates promoters 1 and 2 in trans, leading to TFAP2C-mediated transcriptional induction of HER2 expression in breast cancer samples. PMID: 29035388
- ctDNA gene mutation profiles varied among HR/HER2 subtypes of metastatic breast cancer (MBC) patients. Identifying mutations associated with treatment resistance could potentially improve therapy selection for MBC patients who have received multiline treatment. PMID: 29807833
- It was concluded that miR494 inhibited the cancer initiating cells phenotype and reversed resistance to lapatinib by inhibiting FGFR2 in HER2-positive gastric cancer. PMID: 29786108
- HER2 overexpression was evident in nearly 25% of Malaysian patients with locally advanced or metastatic gastric cancer. The overexpression correlated significantly with male gender and diffuse-type tumors. PMID: 28124769
- There was a statistically significant association between positive p95-HER2 expression and negative hormonal receptors expression (p=0.004), high Ki-67 expression (p<0.001) and the development of visceral metastasis. PMID: 29779938
- The authors herein prove, for the first time, that the transcriptional repressor Blimp1 is a novel mediator of p130Cas/ErbB2-mediated invasiveness. High Blimp1 expression levels are detected in invasive p130Cas/ErbB2 cells and correlate with metastatic status in human breast cancer patients. PMID: 28442738
- ERBB2 amplification is a driving force behind resistance to erlotinib in lung adenocarcinoma. PMID: 28870636
- Results indicate that combining the results of IHC and FISH according to the HER2 testing algorithm is a valuable method for accurately evaluating HER2-positive EMPD. PMID: 29744813
- Due to lower concordance rates of HER2 IHC score 2/3+ cases compared to HER2 IHC score 0/1+ cases, further studies are required to develop detailed analysis criteria for HER2 IHC score 2+ or 3+. PMID: 28478639
- HER2 interacts with Beclin 1 in breast cancer cells and inhibits autophagy. Mice with increased basal autophagy due to a genetically engineered mutation in Becn1 are protected from human HER2-driven mammary tumorigenesis. HER2-mediated inhibition of Beclin 1 and autophagy likely contributes to HER2-mediated tumorigenesis. PMID: 29610308
- These findings suggest that early-stage morphological alterations of HER2-positive BC cells during cancer progression can occur in a physical and signaling-independent manner. PMID: 27599456
Q&A
What is the significance of ErbB2-T686 phosphorylation in ErbB2 signaling?
ErbB2-T686 phosphorylation represents one of several phosphorylation sites on the ErbB2 receptor that may influence receptor signaling. Unlike the more extensively studied phosphorylation sites such as Y1248, phosphorylation at T686 appears to maintain relatively higher basal levels in certain breast cancer cell lines such as BT474. Research indicates that this phosphorylation is detectable in BT474 cells even without stimulation, suggesting it may play a role in constitutive signaling . Notably, unlike phosphorylation at Y1248, T686 phosphorylation is not significantly increased following treatment with either trastuzumab or EGF, implying distinct regulatory mechanisms for this site .
How does phosphorylation at T686 differ functionally from other ErbB2 phosphorylation sites?
The functional differences between T686 and other phosphorylation sites lie in their response to stimuli and their roles in downstream signaling:
| Phosphorylation Site | Response to Trastuzumab | Response to EGF | Associated Function |
|---|---|---|---|
| ErbB2-T686 | No significant change | No significant change | Constitutive signaling |
| ErbB2-Y1248 | Increased phosphorylation | Increased in SKBR3, not BT474 | Associated with trastuzumab sensitivity |
| ErbB2-Y1112 | Minimal change | Increased phosphorylation | Associated with c-Cbl binding and ubiquitination |
| ErbB2-S1113 | No significant change | No significant change | Similar to T686, higher basal levels |
While Y1248 phosphorylation couples ErbB2 to the Ras-Raf-MAP kinase pathway and is associated with trastuzumab response, T686 phosphorylation appears to remain relatively stable regardless of receptor activation status in certain cell lines .
What are the optimal experimental controls when studying Phospho-ERBB2 (T686)?
When studying Phospho-ERBB2 (T686), implement these essential controls:
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Total ErbB2 detection: Always run parallel blots or reprobes for total ErbB2 to normalize phosphorylation levels.
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Phosphatase treatment control: Treat lysate samples with lambda phosphatase to confirm antibody specificity for phosphorylated epitopes.
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Positive control samples: Include lysates from BT474 cells, which demonstrate detectable basal levels of T686 phosphorylation .
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Negative control samples: Use cell lines with low/no ErbB2 expression.
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Treatment controls: Include samples treated with ErbB2 kinase inhibitors like lapatinib to establish baseline after phosphorylation blockade .
What cell lines are recommended for studying ErbB2-T686 phosphorylation?
Based on the available research, these cell lines are recommended:
BT474 cells are particularly valuable for T686 phosphorylation studies due to their documented basal phosphorylation levels at this site .
How do different kinase inhibitors affect ErbB2-T686 phosphorylation compared to other phosphorylation sites?
The effects of kinase inhibitors on ErbB2 phosphorylation sites vary significantly, providing insights into the regulatory mechanisms governing each site:
Interestingly, while lapatinib effectively blocks trastuzumab-mediated phosphorylation at ErbB1-Y845, trastuzumab can still induce phosphorylation of ErbB2-Y1248 in the presence of lapatinib, suggesting that some phosphorylation events may be partially independent of ErbB1/ErbB2 kinase activities . This differential response pattern may extend to T686 as well, potentially implicating other kinases in its regulation.
What is the relationship between ErbB2-T686 phosphorylation and resistance to targeted therapies?
While direct evidence specifically connecting T686 phosphorylation to therapeutic resistance is limited, research on ErbB2 phosphorylation in general provides a framework for understanding potential mechanisms:
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Constitutive signaling: Sites with high basal phosphorylation like T686 may contribute to sustained pathway activation despite targeted therapy .
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Alternative pathway activation: Unlike Y1248, which shows increased phosphorylation upon trastuzumab treatment in sensitive cells, constitutively phosphorylated sites may indicate alternative signaling mechanisms not effectively targeted by current therapies .
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Biomarker potential: The relative stability of T686 phosphorylation across treatment conditions suggests it might serve as a consistent biomarker for ErbB2 status, independent of activation state .
Research investigating phosphorylation at ErbB2-Y1248 has shown correlation with trastuzumab sensitivity, suggesting that different phosphorylation sites may have distinct roles in determining therapeutic response .
How does non-receptor tyrosine kinase CHK interact with ErbB2 phosphorylation at different sites?
Research has revealed a novel mechanism involving non-receptor Csk-homologous kinase (CHK) in regulating ErbB2 phosphorylation:
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CHK recruitment: Trastuzumab promotes recruitment of CHK to ErbB2, influencing both phosphorylation and receptor degradation .
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Site-specific effects: Overexpression of CHK mimics trastuzumab treatment, enhancing phosphorylation particularly at Y1248, which may have implications for other sites including T686 .
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Degradation pathway: CHK binding increases ErbB2-Y1248 phosphorylation and promotes ErbB2 degradation, reducing Akt signaling and inhibiting cell growth .
The identified interactions between CHK and ErbB2 suggest a complex regulatory network where different phosphorylation sites may have interdependent effects on receptor stability and signaling capacity.
What are the optimal conditions for detecting ErbB2-T686 phosphorylation in Western blot experiments?
For optimal detection of ErbB2-T686 phosphorylation by Western blotting:
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Sample preparation:
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Harvest cells at 70-80% confluence
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Lyse cells in buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate)
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Maintain samples at 4°C throughout processing
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Gel electrophoresis and transfer:
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Antibody incubation:
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Detection considerations:
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Enhanced chemiluminescence with extended exposure times may be necessary
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Consider using signal amplification systems for low-abundance phosphorylation sites
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How can I design experiments to distinguish between the roles of different ErbB2 phosphorylation sites?
To effectively differentiate the functional roles of various ErbB2 phosphorylation sites:
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Site-directed mutagenesis approach:
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Generate ErbB2 constructs with specific mutations (T686A, Y1248F, etc.)
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Transfect into ErbB2-negative cell lines
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Compare downstream signaling and cellular responses
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Phosphorylation-specific antibody arrays:
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Kinase inhibitor panel:
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Time-course experiments:
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Monitor phosphorylation at different sites following stimulation or inhibition
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Document temporal relationships between phosphorylation events
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This multifaceted approach can help establish the hierarchical relationships between different phosphorylation sites and their respective contributions to ErbB2 signaling.
What approaches are most effective for correlating ErbB2-T686 phosphorylation with clinical outcomes?
To establish correlations between ErbB2-T686 phosphorylation and clinical outcomes:
Research on ErbB2-Y1248 has demonstrated that positive staining in ErbB2-positive breast cancer biopsies correlates with increased trastuzumab response in neoadjuvant settings , suggesting similar approaches could be valuable for investigating T686 phosphorylation.
How do I troubleshoot inconsistent detection of ErbB2-T686 phosphorylation?
When encountering variable results in phospho-T686 detection:
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Sample preparation issues:
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Ensure rapid sample processing to prevent phosphatase activity
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Verify complete protease and phosphatase inhibition
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Standardize cell culture conditions (confluence, serum levels, passage number)
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Antibody-related considerations:
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Technical optimizations:
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For Western blotting, ensure complete transfer of high molecular weight proteins
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For IHC, optimize antigen retrieval conditions specifically for phospho-epitopes
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Consider alternative detection methods (ELISA, phospho-flow cytometry)
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Biological variables:
How do I interpret contradictory results between different detection methods for ErbB2-T686 phosphorylation?
When facing discrepancies between detection methods:
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Consider method-specific limitations:
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Western blotting provides population averages but may miss cell-to-cell variability
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IHC preserves spatial information but may have lower quantitative precision
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Phospho-specific ELISA offers quantification but loses cellular context
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Evaluation framework:
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Validation approaches:
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Use multiple antibodies targeting the same phosphorylation site
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Employ genetic approaches (site-directed mutagenesis) to validate specificity
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Compare results across different experimental systems
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Biological interpretation:
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Consider that different detection methods may reveal different aspects of the same biological process
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Evaluate results in context of known biology and signaling relationships
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What statistical approaches are most appropriate for analyzing ErbB2-T686 phosphorylation data?
For robust statistical analysis of phosphorylation data:
When analyzing kinase activity in response to treatments, researchers have used approaches such as comparing fold-changes relative to controls with appropriate statistical tests (p-values reported for specific comparisons) .
