EID1 Antibody

What is EID1 and what cellular functions does it regulate?

EID1 (EP300 interacting inhibitor of differentiation 1) is a 21 kDa protein that functions as a transcriptional regulator with multiple roles:

• Inhibits EP300 and CBP histone acetyltransferase activity

• Acts as a repressor of MYOD1 transactivation

• Binds to retinoblastoma protein (RB1) and influences cell cycle regulation

• Functions in coupling cell cycle exit to transcriptional activation of differentiation-related genes

• May serve as a coinhibitory factor for NR0B2 (nuclear receptor subfamily 0, group B, member 2)

EID1 is particularly unstable in G0 cells, with a half-life measured in minutes, suggesting its degradation is tightly regulated during cell cycle progression .

What applications are EID1 antibodies most reliable for?

Based on validation studies, EID1 antibodies demonstrate reliability in the following applications:

Note: For optimal results, each antibody should be titrated in your specific experimental system .

What is the observed molecular weight of EID1 in Western blot analyses?

While the calculated molecular weight of EID1 is 21 kDa (from its 187 amino acid sequence), the observed molecular weight in Western blot analyses typically ranges between 20-30 kDa . This discrepancy may be due to post-translational modifications or the inherent properties of the protein affecting its migration in SDS-PAGE.

How does the EID1 degron overlap with functional domains impact experimental design?

The EID1 degron (residues 160-172) that is recognized by the FBXO21 E3 ubiquitin ligase overlaps with:

• The retinoblastoma tumor suppressor protein (pRB)-binding domain

• The melanoma-associated antigen (MAGE)-binding motif

This overlap creates important considerations for experimental design:

• Mutations targeting the degron may inadvertently disrupt binding to pRB or MAGE proteins

• When studying protein-protein interactions, researchers should be aware that binding of pRB or MAGE proteins may shield EID1 from FBXO21 recognition, potentially stabilizing EID1

• To distinguish effects on degradation versus protein-protein interactions, researchers should design targeted mutations and complementary binding assays

• When using fusion proteins, careful consideration should be given to whether the fusion might disrupt degron accessibility

What experimental approaches can verify the SCF complex-mediated regulation of EID1?

To verify SCF complex-mediated regulation of EID1, several complementary approaches have been validated:

• In vitro ubiquitylation assays:

  • Express Myc-tagged FBXO21 (wild-type and F-box deleted versions) in cells

  • Immunoprecipitate with anti-Myc antibodies

  • Add 35S-labeled EID1 produced by in vitro translation

  • Supplement with recombinant E1 and E2 enzymes

  • Analyze ubiquitylation by SDS-PAGE and autoradiography

• In vivo ubiquitylation assays:

  • Co-express T7-EID1 and HA-ubiquitin in cells

  • Treat with proteasome inhibitor MG132

  • Perform immunoprecipitation under denaturing conditions

  • Detect ubiquitylated species by anti-HA immunoblotting

• Chemical inhibition:

  • Treat cells with MLN4924 (a specific inhibitor of NEDD8-activating enzyme)

  • This inhibits all cullin-RING ligases including SCF complexes

  • Monitor EID1 accumulation by immunoblotting

• Genetic manipulation:

  • Knockdown FBXO21 or CUL1 using siRNA, shRNA, or CRISPR-Cas9

  • Measure EID1 protein accumulation

  • Perform cycloheximide chase experiments to assess protein stability

How can researchers effectively study EID1's cell cycle-dependent regulation?

To study EID1's cell cycle-dependent regulation, researchers can employ these methodological approaches:

• Cell cycle synchronization models:

  • Serum starvation of T98G glioblastoma cells or WI-38 fibroblasts (induces G0)

  • T-cell activation models (transition from G0 to cycling)

  • Monitor EID1 protein levels by immunoblotting

  • Compare with EID1 mRNA levels to confirm post-transcriptional regulation

• Bicistronic reporter systems:

  • Utilize a bicistronic reporter encoding EID1-firefly luciferase fusion and renilla luciferase

  • Compare firefly/renilla luciferase ratios across different cell cycle phases

  • This approach separates transcriptional from post-transcriptional regulation

• Genetic manipulation:

  • Knockdown FBXO21 using siRNA or CRISPR-Cas9

  • Examine how loss of FBXO21 affects EID1 levels in both cycling and G0 cells

  • This determines whether the same or different mechanisms regulate EID1 in different cell cycle phases

• Immunofluorescence microscopy:

  • Perform co-staining of EID1 with cell cycle markers

  • Combine with EdU labeling to identify S-phase cells

  • This reveals cell cycle-dependent changes in EID1 localization and abundance

What are the critical technical considerations when studying EID1 degradation?

Several technical considerations are crucial when studying EID1 degradation:

• Proteasome inhibition:

  • Use MG132 (10 μM) to block proteasomal degradation

  • Include time-course experiments as prolonged inhibition can have secondary effects

  • Compare EID1 accumulation in wild-type versus degron mutants (e.g., EID1(164-166AAA) or EID1(1-157))

• Half-life determination:

  • Perform cycloheximide chase experiments (inhibits protein synthesis)

  • Monitor EID1 disappearance by immunoblotting

  • Compare wild-type EID1 to degron mutants

  • Use FBXO21 knockout/knockdown cells as controls

• Control constructs:

  • Include GFP-EID1 fusion constructs to visualize degradation

  • Compare full-length EID1 to truncation mutants lacking C-terminal residues (residues 151-187 contain the degron)

  • Use alanine scanning mutants to precisely map critical residues (L165 is particularly important)

• Cell type considerations:

  • EID1 degradation rates vary significantly between cell types

  • Particularly pronounced differences exist between cycling and quiescent cells

  • Include multiple cell types in your experimental design

How should researchers troubleshoot non-specific binding when using EID1 antibodies?

When encountering non-specific binding with EID1 antibodies, implement these methodological solutions:

• Validation controls:

  • Include lysates from cells with CRISPR-mediated EID1 knockout

  • Use siRNA-mediated EID1 knockdown samples alongside controls

  • Test multiple independent antibodies recognizing different epitopes

• Optimization strategies:

  • For Western blot: Test different blocking agents (5% milk vs. BSA)

  • For IHC: Compare antigen retrieval methods (citrate buffer pH 6.0 vs. TE buffer pH 9.0)

  • Titrate antibody concentrations carefully (1:500-1:1000 for WB; 1:20-1:200 for IHC)

• Sample preparation considerations:

  • Include proteasome inhibitors (MG132) in lysis buffers to prevent degradation

  • Consider analyzing nuclear and cytoplasmic fractions separately

  • For tissues with low EID1 expression, enrich samples via immunoprecipitation before analysis

What controls are essential when studying EID1 and its interacting partners?

When investigating EID1 and its interactions, incorporate these essential controls:

• For protein-protein interaction studies:

  • Include EID1 mutants disrupting specific interactions:

    • EID1(164-166AAA): Disrupts FBXO21 binding

    • EID1(1-157): Lacks the C-terminal degron entirely

    • LXCXE motif mutants: Disrupt pRB binding

  • Test reciprocal co-immunoprecipitations

  • Verify interactions with both overexpressed and endogenous proteins

• For degradation studies:

  • Compare wild-type EID1 to EID1(164-166AAA) and EID1(1-157)

  • Include both FBXO21 and MDM2 knockdowns (previously suggested as an EID1 regulator)

  • Use both siRNA and CRISPR-Cas9 approaches to rule out off-target effects

• For functional studies:

  • Include rescue experiments with wild-type and mutant EID1

  • Consider cell cycle status (cycling vs. quiescent)

  • Compare multiple cell types (fibroblasts, epithelial cells, T-cells)

What methods are most effective for studying EID1's role in transcriptional regulation?

To investigate EID1's transcriptional regulatory functions, implement these methodological approaches:

• Chromatin immunoprecipitation (ChIP) assays:

  • Perform ChIP using antibodies against EID1, p300/CBP, and other interacting partners

  • Compare chromatin binding in wild-type versus EID1-depleted cells

  • Analyze target genes involved in differentiation and cell cycle control

• Reporter gene assays:

  • Use luciferase reporters driven by promoters regulated by EID1 interacting partners

  • Compare effects of wild-type EID1 versus mutants

  • Include combination treatments with proteasome inhibitors to assess stability effects

• Gene expression analysis:

  • Perform RNA-seq in EID1-overexpressing or EID1-depleted cells

  • Analyze expression changes in genes regulated by CREB, p300/CBP, and pRB

  • Compare transcriptomes of cycling versus quiescent cells

• Co-factor competition assays:

  • Test how EID1 affects binding of p300/CBP to other transcription factors

  • Examine whether EID1 competes with histone acetyltransferase co-activators

  • Investigate how EID1 stability affects these competitions

How can researchers distinguish between EID1 and its paralog EID2 in experimental systems?

To distinguish between EID1 and its paralog EID2 in experiments, researchers should:

• Antibody selection:

  • Use antibodies raised against non-conserved regions

  • Validate antibody specificity using overexpression of each paralog

  • Confirm specificity with siRNA knockdown of each paralog separately

• Expression analysis:

  • Perform paralog-specific qRT-PCR

  • Compare expression patterns across different cell types and conditions

  • Note that EID1 and EID2 may show different cell cycle regulation patterns

• Functional discrimination:

  • Design paralog-specific knockdown experiments (siRNA, shRNA, CRISPR-Cas9)

  • Create rescue experiments with one paralog in the background of the other's depletion

  • Examine differential binding to interaction partners like FBXO21, which interacts with both EID1 and EID2

• Stability assessment:

  • Compare half-lives of EID1 and EID2 in the same cellular context

  • Examine whether the same or different E3 ligases regulate their stability

  • Test whether they respond similarly to cell cycle cues

How should researchers interpret variations in EID1 molecular weight across different experimental systems?

When encountering variations in EID1 molecular weight, consider these interpretation guidelines:

• Expected molecular weight profile:

  • Calculated molecular weight: 21 kDa (187 amino acids)

  • Observed range in SDS-PAGE: 20-30 kDa

• Sources of variation:

  • Post-translational modifications: EID1 may be subject to ubiquitination, phosphorylation, or acetylation

  • Polyubiquitinated forms appear as high-molecular-weight smears after proteasome inhibition

  • Different cell types may process EID1 differently

• Technical factors:

  • Gel percentage affects migration patterns (10-15% gels recommended)

  • Running conditions (voltage, time) impact apparent molecular weight

  • Sample preparation methods may affect observation of modified forms

• Verification approaches:

  • Compare migration patterns with recombinant EID1 standard

  • Use epitope-tagged constructs (e.g., GFP-EID1) with known size shifts

  • Perform immunoprecipitation followed by mass spectrometry to identify modifications

How can researchers reconcile contradictory findings about EID1 regulation?

When facing contradictory findings about EID1 regulation, apply these analytical approaches:

What can researchers infer about EID1 function from its degradation patterns?

The degradation patterns of EID1 provide valuable functional insights:

• Cell cycle regulation:

  • EID1 is particularly unstable in G0 cells but stabilized during cell cycle

  • This pattern suggests EID1 functions are primarily needed in cycling cells

  • The protein may help coordinate cell cycle progression with differentiation programs

• Functional domains and protein interactions:

  • The EID1 degron (residues 160-172) overlaps with pRB and MAGE-binding regions

  • This overlap suggests competitive binding may regulate EID1 stability

  • pRB or MAGE proteins might shield EID1 from FBXO21 recognition, stabilizing EID1 during specific cellular processes

• Regulatory logic:

  • Rapid degradation enables tight temporal control of EID1 functions

  • The protein likely mediates transient signaling events rather than structural roles

  • Its instability in G0 suggests it may be needed to exit quiescence but not maintain it

• Disease implications:

  • Dysregulated EID1 degradation could impact cell cycle control and differentiation

  • Understanding how mutations affect EID1 stability may provide insights into disease mechanisms

  • The protein's role in acetylation-dependent gene regulation connects it to multiple cellular pathways

What are the optimal conditions for immunohistochemical detection of EID1 in tissue samples?

For optimal immunohistochemical detection of EID1, follow these methodological guidelines:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval (pressure cooker or microwave) typically yields better results than enzymatic methods

• Antibody dilution and incubation:

  • Recommended dilution range: 1:20-1:200

  • Optimal dilution requires titration for each tissue type

  • Overnight incubation at 4°C often yields better signal-to-noise ratio than shorter incubations

• Detection systems:

  • Polymer-based detection systems generally provide better sensitivity than avidin-biotin methods

  • For tissues with low EID1 expression, consider tyramide signal amplification

  • Include appropriate negative controls (isotype control antibodies and EID1-negative tissues)

• Sample considerations:

  • EID1 has been successfully detected in human breast cancer tissue

  • Expression varies by cell cycle status, so interpretation should consider proliferative state

  • Nuclear staining is expected based on EID1's function, but cytoplasmic staining may also be observed

How should researchers design experiments to study EID1's role in differentiation processes?

To investigate EID1's role in differentiation, implement these experimental design strategies:

• Differentiation model systems:

  • Myogenic differentiation: C2C12 cells (EID1 represses MyoD1 transactivation)

  • Adipogenic differentiation: 3T3-L1 cells

  • Neuronal differentiation: SH-SY5Y or primary neuronal cultures

  • Monitor EID1 levels throughout differentiation timeline

• Gain and loss of function approaches:

  • Generate stable cell lines with inducible EID1 expression

  • Create EID1 knockdown or knockout cells using RNAi or CRISPR-Cas9

  • Rescue experiments with wild-type and mutant EID1 (particularly p300-binding and degron mutants)

• Molecular readouts:

  • Assess histone acetylation status at differentiation-specific promoters

  • Monitor expression of differentiation markers by qRT-PCR and Western blotting

  • Perform ChIP assays to examine p300/CBP recruitment to target genes

• Functional assays:

  • Morphological assessment of differentiation

  • Biochemical markers of terminal differentiation

  • Cell cycle exit analysis (EID1 may couple cell cycle exit to differentiation)

What approaches can verify EID1's role in the structural maintenance of chromosomes (SMC) complex?

To investigate EID1's proposed role in SMC complexes, researchers should:

• Interaction verification:

  • Perform co-immunoprecipitation of EID1 with SMC complex components

  • Test interaction with MAGE proteins (proposed mammalian counterparts of yeast Nse3)

  • Map interaction domains through truncation and point mutations

• Functional assays:

  • DNA damage response assays in EID1-depleted cells

  • Sister chromatid exchange frequency measurement

  • Metaphase spread analysis for chromosomal abnormalities

  • Comet assays to detect DNA breaks

• Localization studies:

  • Immunofluorescence co-localization of EID1 with SMC complex proteins

  • Analysis of EID1 recruitment to DNA damage sites

  • Chromatin fractionation to detect EID1 association with chromatin

• Cell cycle-dependent regulation:

  • Compare EID1 association with SMC components across cell cycle

  • Examine whether MAGE protein binding protects EID1 from degradation

  • Test if DNA damage alters EID1 stability or SMC complex association