SLC22A1 Antibody, FITC conjugated
Description
SLC22A1 Function
SLC22A1 (OCT1) is a 12-transmembrane domain protein primarily expressed in hepatocytes, enterocytes, and renal proximal tubules. It mediates the bidirectional transport of endogenous small organic cations (e.g., acetylcholine, creatinine) and a wide array of drugs (antimalarials, metformin, tyrosine kinase inhibitors) . Genetic polymorphisms (e.g., SLC22A1 1022C>T) reduce OCT1 activity, altering drug pharmacokinetics and efficacy .
FITC Conjugation
FITC is a fluorescent dye with excitation/emission wavelengths of 495 nm/520 nm. Conjugation to antibodies enhances detection sensitivity in:
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Flow cytometry: Quantifying SLC22A1 surface expression on cells.
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Immunofluorescence microscopy: Localizing OCT1 in tissues or cell cultures.
Drug Transport Studies
SLC22A1 antibodies are pivotal in probing OCT1’s role in pharmacokinetics:
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Proguanil Metabolism: SLC22A1 1022C>T polymorphism reduces proguanil uptake into hepatocytes, lowering cycloguanil (active metabolite) levels . FITC-conjugated antibodies could track OCT1 localization in liver cells to validate these findings.
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Metformin Uptake: Structural studies reveal hOCT1’s conformational flexibility during metformin transport . Fluorescent antibodies could monitor transporter dynamics in real-time.
Cancer Research
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Hepatocellular Carcinoma (HCC): OCT1 expression correlates with sorafenib response in HCC patients . FITC-conjugated antibodies enable imaging OCT1 in tumor biopsies to assess therapeutic potential.
Metabolomics
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Acylcarnitine Efflux: SLC22A1 exports acylcarnitines from hepatocytes, linking its activity to metabolic diseases . Fluorescent antibodies aid in visualizing transporter localization during acylcarnitine efflux assays.
Pharmacogenomic Impact
Challenges and Future Directions
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Polymorphism Impact: SLC22A1 variants (e.g., 1022C>T) require genotype-specific antibody validation for accurate detection.
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Cross-Reactivity: Ensure antibodies distinguish SLC22A1 from paralogs (e.g., SLC22A2, SLC22A3) .
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Therapeutic Monitoring: FITC-conjugated antibodies may enable real-time tracking of OCT1 in personalized medicine (e.g., drug response prediction).
References and Resources
Product Specs
Target Background
This antibody targets Organic Cation Transporter 1 (OCT1), encoded by the SLC22A1 gene. OCT1 is a membrane transporter protein that facilitates the bidirectional translocation of a wide range of organic cations across the plasma membrane. These substrates include various model compounds (e.g., 1-methyl-4-phenylpyridinium (MPP+), tetraethylammonium (TEA), N-1-methylnicotinamide (NMN), 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP)), endogenous compounds (e.g., choline, guanidine, histamine, epinephrine, adrenaline, noradrenaline, dopamine), and pharmaceuticals (e.g., quinine, metformin). Transport activity is modulated by numerous inhibitors, including tetramethylammonium (TMA), cocaine, lidocaine, NMDA receptor antagonists, atropine, prazosin, cimetidine, TEA, NMN, guanidine, choline, procainamide, quinine, tetrabutylammonium, and tetrapentylammonium. OCT1 functions in an electrogenic and pH-independent manner. Furthermore, it transports polyamines such as spermine and spermidine, and facilitates the basolateral membrane transport of pramipexole in proximal tubular epithelial cells. Choline transport is notably activated by MMTS. Regulation of OCT1 activity is complex, involving intracellular signaling pathways, including inhibition by protein kinase A activation and endogenous activation via the calmodulin complex, calmodulin-dependent kinase II, and LCK tyrosine kinase.
Numerous studies have investigated the role of SLC22A1 genetic variations in drug metabolism and disease:
- Genetic variations in SLC22A1 exon 7 (SNPs and indels) have been associated with imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) patients. (PMID: 30262695)
- OCT1 contributes to metformin uptake and regulates pancreatic stellate cell activity. (PMID: 29949790)
- The OCT1*2 allele influences the inhibitory potency of morphine uptake, while OCT2 ranitidine uptake is less affected by the Ala270Ser polymorphism. (PMID: 29236753)
- OCT1 rs628031 and ABCG2 rs2231142 polymorphisms are associated with plasma lamotrigine concentrations in Han Chinese epilepsy patients. (PMID: 27610747)
- SLC22A1/OCT1 genetics play a role in neonatal exposure to M1. (PMID: 27082504)
- The pregnane X receptor downregulates OCT1 in human hepatocytes by competing for SRC-1 coactivator. (PMID: 26920453)
- SLC22A1 gene variants are associated with serum acylcarnitines and metabolic diseases. (PMID: 28942964)
- Research has explored the hepatic gene regulation of human OCT1, including potential post-transcriptional regulation by miRNAs. (PMID: 27278216)
- Metformin treatment response (HbA1c, HOMA-IR, fasting insulin, glucose changes) did not differ significantly between SLC22A1 wild-type and low-activity allele carriers. (PMID: 27407018)
- miR-21 silencing increased chemosensitivity to various drugs and upregulated expression of SLC22A1/OCT1, SLC22A2/OCT2, and SLC31A1/CTR1 in renal cell carcinoma. (PMID: 28714373)
- Stilbenoid treatment alters the chromatin structure of the MAML2 enhancer, affecting OCT1 transcription factor occupancy and MAML2 methylation. (PMID: 27207652)
- OCT1 genetic variations (R61C, M420del) are associated with metformin intolerance in type 2 diabetes patients. (PMID: 26605869)
- Intratumoral OCT1 mRNA expression shows promise as a prognostic biomarker in hepatocellular carcinoma (HCC). (PMID: 26872727)
- Homozygous OCT1 C-allele carriers had lower rates of metformin-related toxicity compared to wild-type A-allele carriers. (PMID: 25753371)
- hOCT1 influences bendamustine cytotoxicity based on expressed genetic variants. (PMID: 25582574)
- No association was found between ABCB1, ABCG2, and OCT1 polymorphisms and thrombocytopenia. (PMID: 26546461)
- Each organic cation transporter (OCT1-3) demonstrates specific involvement in drug transport. (PMID: 25883089)
- OCT1 exon 2 GG homozygotes showed higher imatinib levels than CG/CC genotypes, although not statistically significant. (PMID: 24524306)
- OCT1 and ABCC3 genotypes, along with body weight, significantly influence intravenous morphine pharmacokinetics in children. (PMID: 25155932)
- Nucleoside transporters and hOCT1 influence the cellular handling of DNA-methyltransferase inhibitors. (PMID: 24780098)
- hOCT1 expression level predicts response to imatinib in CML patients. (PMID: 25358338)
- OCT1 plays a significant role in hepatic serotonin elimination. (PMID: 24688079)
- Clopidogrel and its metabolite are strong inhibitors and high-affinity substrates of OCT1. (PMID: 24530383)
- OCT1 genetic variants are associated with long-term outcomes in imatinib-treated CML patients. (PMID: 24215657)
- OCT1 genetic polymorphisms are associated with primary biliary cirrhosis development and progression. (PMID: 23612856)
- Imatinib cellular uptake is independent of OCT1, suggesting it is not a valid biomarker for imatinib resistance. (PMID: 24352644)
- OCT1, OCT2, and ATM variants are associated with elevated C-peptide levels in polycystic ovary syndrome. (PMID: 24533710)
- Rhodamine 123 is a high-affinity substrate for both hOCT1 and hOCT2. (PMID: 22913740)
- Decreased SLC22A1 mRNA expression is associated with poor imatinib response in CML. (PMID: 24469953)
- Glucocorticoid receptor-induced HNF4α expression may upregulate OCT1 indirectly. (PMID: 24399729)
- OCT1 genotypes significantly impact intravenous morphine pharmacokinetics. (PMID: 23859569)
- Hepatocellular and cholangiocarcinoma show OCT1 downregulation and genetic variants affecting sorafenib response. (PMID: 23532667)
- The SNP 408V>M (g.1222G>A) is associated with an 8-base-pair insertion in CML patients, impacting treatment outcomes. (PMID: 24117365)
- Oct1 mRNA expression is mediated by loss of T cells in immune-mediated liver disease. (PMID: 23929842)
- The intron 1 evolutionary conserved region of OCT1 increases Oct1 promoter activity. (PMID: 23922447)
- Lamivudine accumulation in CD4 cells of HIV-infected patients is related to OCT1 and OCT2 expression. (PMID: 22875535)
- hOCT1 likely plays a role in the disposition of fluoroquinolone antimicrobial agents. (PMID: 23545524)
- PER2 acts as a transcriptional corepressor, recruiting proteins to OCT1 (POU2F1) binding sites on the TWIST1 and SLUG promoters. (PMID: 23836662)
- OCT1 downregulation is associated with tumor progression and poorer patient survival. (PMID: 23440379)
- OCT1 -1756 genotypes do not affect expression levels. (PMID: 22498645)
- SLC22A1-ABCB1 haplotypes may influence imatinib pharmacokinetics in Asian CML patients. (PMID: 23272163)
- Structural requirements for OCT1 and OCT2 are discussed and compared to the BBB choline transporter. (PMID: 22483271)
- Oct1 regulates normal and cancer stem cell function. (PMID: 23144633)
- Seven polymorphisms in OCT1, OCT2, and MATE1 genes were compared in type 2 diabetes patients with and without metformin side effects. (PMID: 22735389)
- The OCT1 SNPs M420del and M408V affect imatinib uptake, with M420del modifying clinical outcome in CML. (PMID: 23223357)
- High-dose imatinib provides better molecular responses in patients with low OCT-1 activity. (PMID: 22207690)
- A substrate binding hinge domain is critical for OCT1 transport-related structural changes. (PMID: 22810231)
- A model for synergistic gene regulation by Sox2 and Oct1 is proposed. (PMID: 22718759)
- hOCT1 gene expression levels were evaluated in CML patients to assess their correlation with imatinib response. (PMID: 22508387)
Q&A
What is SLC22A1 and why is it important in research?
SLC22A1, also known as organic cation transporter 1 (OCT1), is a plasma membrane transporter primarily expressed in the liver. It belongs to the solute carrier family 22 and plays a crucial role in the transport of various endogenous and pharmacological molecules between the liver and blood. In humans, the canonical protein has a reported length of 554 amino acid residues with a molecular weight of approximately 61.2 kDa . Recent research has uncovered SLC22A1's significant role in acylcarnitine efflux from the liver to circulation, making it an important target for metabolic disease research .
The protein is localized to the hepatocyte basolateral (sinusoidal) membrane and transports substances containing quaternary amine groups, a property shared by carnitine and acylcarnitines . SLC22A1 is important in research for several reasons: it serves as a model for understanding membrane transport mechanisms, its role in drug disposition makes it relevant for pharmacokinetic studies, and its associations with serum acylcarnitine levels connect it to metabolic pathways. Additionally, genetic variations in SLC22A1 have been linked to various disease states and drug responses, as evidenced by genome-wide association studies identifying a signal at the SLC22A1 locus for serum acylcarnitines .
What are the key applications for FITC-conjugated SLC22A1 antibodies?
FITC-conjugated SLC22A1 antibodies offer several advantages for research applications due to the direct fluorescent labeling. The key applications include:
For optimal results, researchers should consider the specificity of the antibody clone, potential cross-reactivity with other organic cation transporters, and the FITC:antibody ratio for signal intensity. FITC-conjugated SLC22A1 antibodies have been validated for applications including ELISA and FACS, making them versatile tools for transporter research .
How does FITC conjugation affect SLC22A1 antibody performance?
FITC (Fluorescein isothiocyanate) conjugation introduces important considerations for SLC22A1 antibody performance that researchers must account for in experimental design:
| Parameter | Effect of FITC Conjugation | Practical Considerations |
|---|---|---|
| Epitope Recognition | May affect binding if conjugation occurs near antigen-binding region | Verify antibody performance post-conjugation |
| Signal Stability | FITC is susceptible to photobleaching | Minimize light exposure, use antifade mounting media |
| pH Sensitivity | FITC fluorescence optimal at pH 8.0, decreases at lower pH | Use alkaline buffers when possible, avoid acidic conditions |
| Fluorescence Properties | Excitation/emission at 495/519 nm (green fluorescence) | Consider spectral overlap when designing multiplex experiments |
| Direct Detection | Eliminates need for secondary antibodies | Reduces background from non-specific secondary binding |
| Quantitative Applications | F:P ratio affects signal intensity | Verify batch consistency for comparative studies |
While FITC conjugation offers the advantage of direct detection, researchers should be aware that the conjugation process may alter the antibody's binding characteristics compared to unconjugated versions. When selecting FITC-conjugated SLC22A1 antibodies, consider clones that have been specifically validated post-conjugation for the intended application, especially for quantitative studies examining transporter expression levels .
What controls should be used when working with FITC-conjugated SLC22A1 antibodies?
When working with FITC-conjugated SLC22A1 antibodies, implementing appropriate controls is crucial for experimental validity:
| Control Type | Description | Purpose |
|---|---|---|
| Isotype Control | FITC-conjugated antibody of same isotype and host species with irrelevant specificity | Distinguishes specific binding from Fc receptor binding or non-specific interactions |
| Blocking Controls | Pre-incubation with immunizing peptide or recombinant SLC22A1 | Confirms antibody specificity |
| Positive Control Samples | Liver tissue or hepatocyte cell lines | Confirms antibody functionality with known high-expression samples |
| Negative Control Samples | Tissues/cells with confirmed absence of SLC22A1 expression | Establishes background signal baseline |
| Autofluorescence Control | Unstained samples | Identifies natural tissue autofluorescence, especially important in liver tissues |
| Secondary-Only Control | When using amplification steps | Identifies non-specific binding of secondary reagents |
| Signal Specificity Validation | Alternative detection methods or antibodies targeting different epitopes | Confirms signal represents true SLC22A1 localization |
For flow cytometry applications, fluorescence-minus-one (FMO) controls help establish proper gating strategies by accounting for spectral overlap when multiple fluorophores are used. These controls are particularly important when examining SLC22A1 expression across different cell populations or experimental conditions to ensure reliable quantitative comparisons .
What is the optimal fixation method for immunofluorescence with FITC-conjugated SLC22A1 antibodies?
The optimal fixation method for immunofluorescence using FITC-conjugated SLC22A1 antibodies should preserve both the antigen epitope integrity and the membrane localization of SLC22A1:
| Fixation Method | Protocol | Advantages/Considerations |
|---|---|---|
| Paraformaldehyde Fixation | 4% PFA, 10-15 min, room temperature | Preserves membrane architecture, maintains epitope accessibility for most clones |
| Methanol Fixation | 100% methanol, 10 min, -20°C | Preserves different epitopes than PFA, but may disrupt membrane structures |
| Gentle Permeabilization | 0.1-0.2% Triton X-100 or 0.1% saponin | Enables antibody access while preserving membrane protein localization |
| Epitope Retrieval | Citrate buffer (pH 6.0), gentle heat | May improve epitope accessibility in heavily fixed samples |
| Buffer Selection | PBS with pH 7.4-8.0 | Enhances FITC fluorescence intensity, which is optimal at slightly alkaline pH |
| Mounting Media | Anti-fade media with DAPI | Reduces photobleaching while providing nuclear counterstaining |
For liver tissue specifically, which is the primary expression site of SLC22A1 , researchers should consider additional steps to reduce the significant autofluorescence often observed. Pre-treatment with Sudan Black B (0.1-0.3%) or brief sodium borohydride incubation can significantly improve signal-to-noise ratio. Optimization through parallel comparison of multiple fixation protocols using positive control samples is recommended to determine the best approach for specific experimental conditions and antibody clones.
How can FITC-conjugated SLC22A1 antibodies be used to investigate acylcarnitine transport mechanisms?
FITC-conjugated SLC22A1 antibodies offer valuable tools for investigating acylcarnitine transport mechanisms, particularly given the recently discovered role of SLC22A1 in acylcarnitine efflux from liver to circulation :
| Research Approach | Methodology | Data Output |
|---|---|---|
| Colocalization Studies | Confocal microscopy with SLC22A1 and acylcarnitine machinery markers | Spatial relationships between transport components |
| Transport Activity Correlation | Correlate FITC signal intensity with isotope tracing data | Structure-function relationships of transporter variants |
| Trafficking Studies | Live-cell tracking of SLC22A1 redistribution | Dynamic response to metabolic challenges |
| Mutational Analysis | Compare localization patterns of wild-type vs. mutant SLC22A1 | Impact of genetic variants on transporter function |
| Metabolomic Integration | Combine imaging with targeted acylcarnitine metabolomics | Correlation between expression and metabolite profiles |
Recent research has demonstrated that SLC22A1 plays a significant role in the efflux of acylcarnitines from the liver to the circulation, with genetic variants affecting this function . By combining FITC-conjugated SLC22A1 antibody-based imaging with isotope tracing experiments ([3H]-L-carnitine labeling), researchers can track both the expression/localization of the transporter and its functional activity in transporting acylcarnitines. This integrated approach has been valuable in validating the impacts of human SLC22A1 variants on acylcarnitine efflux in vitro, explaining their association with serum acylcarnitine levels .
What are the considerations for multiplexing FITC-conjugated SLC22A1 antibodies with other fluorescent markers?
Multiplexing FITC-conjugated SLC22A1 antibodies with other fluorescent markers requires careful planning to achieve optimal signal separation and data quality:
| Consideration | Technical Approach | Recommended Parameters |
|---|---|---|
| Spectral Overlap Management | Select spectrally distant fluorophores | Pair FITC with far-red dyes (Alexa 647), deep red (Alexa 700), or blue (DAPI) |
| Compensation Requirements | Generate proper compensation matrix | Include single-stained controls for each fluorophore |
| Sequential Imaging Strategy | Capture channels separately for overlapping fluorophores | Minimizes bleed-through for microscopy applications |
| Filter Selection | Use narrow bandpass emission filters | 515-535 nm for FITC provides better signal specificity |
| Photobleaching Management | Capture FITC signals early in acquisition sequence | Compensates for FITC's relatively rapid photobleaching |
Recommended Multiplex Combinations:
| Application | Fluorophore Combination | Target Combinations |
|---|---|---|
| 3-Color Imaging | DAPI/FITC/Alexa 647 | Nuclei/SLC22A1/Organelle marker |
| 4-Color Imaging | DAPI/FITC/Alexa 555/Alexa 647 | Nuclei/SLC22A1/Interacting protein/Organelle marker |
| Flow Cytometry | FITC/PE-Cy7/APC | SLC22A1/Surface marker/Viability dye |
For liver tissue specifically, additional autofluorescence quenching steps are recommended due to the high natural fluorescence in this tissue type. When studying transporter interactions or trafficking pathways, strategic combinations of markers can reveal relationships between SLC22A1 and other cellular components involved in acylcarnitine transport or drug disposition .
How can FITC-conjugated SLC22A1 antibodies be used to distinguish between different isoforms?
Distinguishing between SLC22A1 isoforms using FITC-conjugated antibodies requires strategic approaches focused on epitope specificity and complementary techniques:
| Strategy | Methodology | Analytical Approach |
|---|---|---|
| Epitope-Specific Antibodies | Select antibodies targeting isoform-specific regions | Compare binding patterns across samples expressing different isoforms |
| Comparative Panel Analysis | Use multiple antibodies targeting distinct epitopes | Create "isoform signatures" based on binding patterns |
| Co-staining Approach | Combine pan-SLC22A1 with isoform-specific antibodies | Visualize relative distribution of specific isoforms |
| Quantitative Flow Cytometry | Measure binding intensity ratios | Detect shifts in isoform expression patterns |
Up to four different isoforms have been reported for human SLC22A1 , which differ in specific amino acid sequences and potentially in their functional characteristics. When investigating isoform distribution, researchers should:
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Verify antibody specificity against known isoforms using overexpression systems
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Compare staining patterns with results from isoform-specific RT-PCR analysis
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Assess potential differences in subcellular localization between isoforms using high-resolution confocal microscopy
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Correlate isoform expression patterns with functional transport assays
This multi-parameter approach provides more reliable isoform characterization than using antibody-based detection alone. For rigorous isoform distinction, antibody-based methods should be complemented with molecular techniques targeting the specific sequence variations that define each isoform .
What are the strategies for optimizing signal-to-noise ratio when using FITC-conjugated SLC22A1 antibodies in liver tissue samples?
Liver tissue presents particular challenges for immunofluorescence due to high autofluorescence and complex architecture. Optimizing signal-to-noise ratio for FITC-conjugated SLC22A1 antibodies in liver samples requires:
| Challenge | Optimization Strategy | Protocol Recommendations |
|---|---|---|
| Tissue Autofluorescence | Chemical quenching treatments | 0.1-0.3% Sudan Black B (10 min) or 0.1% sodium borohydride (5-10 min) |
| Tissue Section Parameters | Optimize section thickness and preparation | 4-5 μm sections; fresh frozen often better than FFPE for membrane proteins |
| Blocking Enhancements | Extended blocking with optimized buffers | 1-2 hours with 5-10% serum plus 0.1-0.3% Triton X-100 |
| Antibody Incubation | Titration and incubation optimization | Extended incubation (overnight at 4°C) with precisely titrated antibody concentration |
| Imaging Approaches | Advanced microscopy techniques | Confocal with narrow bandpass filters; spectral imaging; deconvolution |
Optimization Workflow:
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Prepare multiple liver tissue sections using identical collection/fixation methods
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Divide sections into test groups for different autofluorescence reduction treatments
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Perform antibody titration series (typically 1:50 to 1:1000 dilutions)
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Compare signal-to-noise ratios across treatment combinations
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Validate specificity using peptide competition and SLC22A1-knockout controls
Given that SLC22A1 is primarily expressed in the liver , optimizing detection in this tissue type is particularly important for physiologically relevant studies. The basolateral (sinusoidal) membrane localization of SLC22A1 should be clearly visible in properly optimized samples, providing a useful internal validation of staining specificity .
How can FITC-conjugated SLC22A1 antibodies be used to investigate post-translational modifications of the transporter?
FITC-conjugated SLC22A1 antibodies can be valuable tools for investigating post-translational modifications (PTMs) when used in strategic combinations with other reagents:
| Research Approach | Methodology | Data Interpretation |
|---|---|---|
| Co-localization with PTM-Specific Antibodies | Multiplex FITC-SLC22A1 with PTM-specific antibodies | Reveals proportion of SLC22A1 carrying specific modifications |
| Differential Detection | Compare pan-SLC22A1 vs. modification-specific antibodies | Distinguishes modified from unmodified transporters |
| PTM-Dependent Trafficking | Track SLC22A1 signals following PTM-modifying treatments | Links modifications to protein trafficking |
| Quantitative Analysis | Measure FITC signal changes after PTM manipulation | Establishes relationships between modifications and protein stability |
SLC22A1 undergoes several post-translational modifications including phosphorylation and glycosylation , which can affect its localization, stability, and transport function. To investigate these modifications:
Experimental Design Table:
| PTM Type | Treatment Approach | Expected Outcome | Control Validation |
|---|---|---|---|
| Phosphorylation | Phosphatase inhibitors (e.g., okadaic acid) | Increased phospho-SLC22A1 | Western blot with phospho-specific antibodies |
| Phosphorylation | Kinase inhibitors (e.g., staurosporine) | Decreased phospho-SLC22A1 | Mass spectrometry validation of sites |
| Glycosylation | Tunicamycin treatment | Blocks new N-glycosylation | PNGase F treatment as control |
| Glycosylation | Swainsonine treatment | Affects complex glycan structure | Lectin staining verification |
By combining imaging approaches with biochemical validation, researchers can establish connections between specific modifications, transporter localization, and functional activity. This is particularly relevant for understanding how genetic variants affecting modification sites might impact SLC22A1's role in acylcarnitine transport and drug disposition .
