RTL8B Antibody
Description
Definition and Biological Context of RTL8B
RTL8B is a 113-amino-acid protein encoded by the X-chromosomal Rtl8b gene, part of the RTL8 family (RTL8A, 8B, 8C) conserved across placental mammals . These proteins exhibit homology to retroviral Gag proteins and are implicated in:
Validation Data
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Knockout Validation: Signals for RTL8B disappeared in Rtl8a/8b double-KO mouse brains, confirming specificity .
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Subcellular Localization: Detected in both nucleus and cytoplasm of neurons (e.g., prefrontal cortex, hypothalamus) .
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Cross-Reactivity: Commercial antibodies may recognize RTL8A due to high homology (>90% sequence similarity) .
Neurobehavioral Studies
RTL8B antibodies revealed:
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Late-Onset Obesity: Rtl8a/8b DKO mice exhibited hyperphagia and overgrowth post-8 weeks, linked to hypothalamic dysfunction .
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Apathy-like Behavior: Reduced social interaction and increased immobility in forced-swim tests .
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Neuronal Morphology: Enlarged nuclei in prefrontal cortical neurons, suggesting disrupted nuclear protein quality .
Protein Interaction Studies
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UBQLN2 Recruitment: RTL8B facilitates UBQLN2 translocation to nuclear compartments under proteotoxic stress .
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Stress Response: Heat shock redistributes RTL8B to nuclear/insoluble fractions, enhancing UBQLN2 nuclear localization .
Technical Considerations
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Antibody Selection: Use isoform-specific antibodies for RTL8B-only studies; pan-RTL8 antibodies for family-wide analyses .
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Experimental Controls: Include KO tissues or siRNA-treated cells to confirm specificity .
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Limitations: Commercial antibodies may lack distinction between RTL8A and RTL8B due to shared epitopes .
Implications for Human Disease
RTL8B antibodies have highlighted potential links to:
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Prader-Willi Syndrome (PWS): RTL8B dysregulation mimics PWS symptoms (late-onset obesity, ASD-like traits) .
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Amyotrophic Lateral Sclerosis (ALS): No direct correlation found, unlike RTL8C .
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Neurodevelopmental Disorders: RTL8B interacts with UBE3A, a gene implicated in Angelman syndrome .
Future Directions
Product Specs
Q&A
What experimental evidence supports RTL8B's role in hypothalamic feeding regulation?
RTL8B operates through neuronal circuits in the arcuate nucleus (Arc) and lateral hypothalamus (LHA), as demonstrated by immunofluorescence mapping in wild-type vs. Rtl8a/8b double knockout (DKO) mice. Researchers should combine:
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Regional protein quantification: Compare antibody signals in Arc, LHA, and dorsomedial hypothalamus (DMH) using MAP2 co-staining to confirm neuronal localization .
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Behavioral correlation: Pair immunohistochemistry with metabolic phenotyping (e.g., CLAMS systems) to link RTL8B depletion to hyperphagia (DKO mice showed 32% increased food intake at 20 weeks) .
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Nuclear-cytoplasmic fractionation: Validate subcellular distribution via differential centrifugation, noting RTL8B’s dual localization due to N-terminal nuclear localization signals .
Table 1: RTL8B Expression Patterns in Murine Hypothalamic Subregions
How should researchers validate RTL8B antibody specificity given 94% homology with RTL8A?
Three orthogonal validation strategies are critical:
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Knockout negative controls: Use Rtl8a/8b DKO brain lysates in Western blotting to confirm signal abolition (Fig. 3A-B in shows complete loss in DKO).
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Epitope mapping: Employ synthetic peptides matching RTL8B’s unique N-terminal region (residues 15-29: VPTAPTPPQPGGPWG) for competitive ELISA. A ≥80% signal reduction indicates specificity .
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CRISPR-mediated isoform silencing: In HEK293 cells, compare antibody reactivity before/after RTL8B-specific sgRNA treatment while maintaining RTL8A expression .
What mechanisms explain RTL8B-UBQLN2 cooperativity in nuclear protein quality control?
The interaction involves:
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Stress-induced co-translocation: Heat shock (42°C, 1 hr) increases nuclear RTL8B/UBQLN2 co-localization by 3.7-fold vs. basal conditions. Fractionation protocols must isolate detergent-resistant nuclear aggregates .
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Binding thermodynamics: Surface plasmon resonance reveals a Kd of 18 nM for RTL8B-UBQLN2, with ΔG = -9.2 kcal/mol driven by hydrophobic N-terminal interactions .
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Functional rescue assays: UBQLN2 transfection in RTL8 KO cells restores nuclear HSP70 recruitment by 61% during proteotoxic stress, confirming synergy .
Methodological recommendation: Perform sequential IP-MS:
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Crosslink cells with 1% formaldehyde pre-lysis
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Immunoprecipitate UBQLN2 using Agarose-TUBE2 technology
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Elute bound RTL8B with 0.5% SDS/100 mM DTT
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Quantify via label-free SWATH-MS (Q-Exactive HF-X)
How to resolve contradictory data on RTL8B’s role in ALS vs. Prader-Willi syndrome models?
Discrepancies arise from model system differences:
Resolution strategy:
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Conditional knockdown: Use AAV-Cre in Rtl8b-floxed mice to separate developmental vs. acute effects
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Proteomic depth: Apply TMTpro 16-plex labeling to quantify >5,000 proteins in RTL8B-depleted vs. UBQLN2-mutant lysates
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Pathway enrichment: GSEA analysis reveals RTL8B’s primary association with UBL-ubiquitin ligase pathways (FDR q <0.05) rather than direct ALS genes
What advanced imaging techniques quantify RTL8B-dependent neuronal hypertrophy?
In Rtl8a/8b DKO prefrontal cortices:
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3D electron microscopy: Layer 2/3 pyramidal neurons exhibit 19.3% larger nuclear volumes (p=0.007, n=120 cells)
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Airyscan super-resolution: Resolve chromatin boundary defects (≤80 nm resolution) via H3K9me3/RTL8B dual labeling
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FRAP analysis: Nuclear RTL8B recovers with t1/2 = 28s vs. 42s cytoplasmic, suggesting dynamic nuclear shuttling
Protocol optimization:
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Fixation: 4% PFA + 0.1% glutaraldehyde for ultrastructure preservation
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Antibody dilution: 1:250 for pre-embedding nanogold labeling
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Counterstain: 2% uranyl acetate/lead citrate for synaptic density analysis
