HRD1A Antibody

What is HRD1 and why is it important to study?

HRD1 is an E3 ubiquitin-protein ligase that forms a critical component of the endoplasmic reticulum quality control (ERQC) system, also called ER-associated degradation (ERAD) . It accepts ubiquitin specifically from endoplasmic reticulum-associated UBC7 E2 ligase and transfers it to substrates, promoting their degradation . HRD1 is important to study because it plays multifaceted roles in cellular homeostasis, including:

• Regulation of misfolded protein degradation in the ER

• Protection against ER stress-induced apoptosis

• Metabolic regulation in the liver

• Immune cell development and function

• Antigen presentation processes

HRD1 dysregulation has been implicated in various pathological conditions, making it a valuable research target for understanding disease mechanisms .

What applications are HRD1 antibodies suitable for?

Based on validated research, HRD1 antibodies are suitable for multiple applications in molecular and cellular biology research:

• Western blotting (WB) for protein expression analysis

• Immunoprecipitation (IP) for studying protein interactions

• Immunocytochemistry/Immunofluorescence (ICC/IF) for localization studies

• Flow cytometry for intracellular staining

• Immunohistochemistry for tissue analysis

When selecting an HRD1 antibody, researchers should verify species reactivity (commonly available for human, mouse, and rat samples) and validate the antibody for their specific application and experimental system .

How do I optimize western blotting conditions for HRD1 detection?

For optimal western blot detection of HRD1:

• Sample preparation: Use cell lysates from HRD1-expressing cells (validated sources include SH-SY5Y, 293T, Ramos, HeLa, and HepG2 cells)

• Loading amount: Start with 10-20 μg of total protein lysate per lane

• Antibody dilution: Use a 1:1000 dilution of primary HRD1 antibody (optimize as needed)

• Detection method: ECL (enhanced chemiluminescence) provides good sensitivity

• Expected band size: Approximately 67 kDa

For validation, include positive controls like HepG2 liver cells, which show consistent HRD1 expression, and consider including HRD1-knockout samples as negative controls when available.

How can I study the interactions between HRD1 and its metabolic substrates?

To investigate HRD1 interactions with metabolic substrates:

• Co-immunoprecipitation (Co-IP): Use anti-HRD1 antibodies for immunoprecipitation followed by immunoblotting for suspected interaction partners. This approach has successfully identified interactions between HRD1 and metabolic regulators including ENTPD5, CPT2, RMND1, HSD17B4, and ATP5D .

• Affinity purification coupled with mass spectrometry (AP-MS): This comprehensive approach identified 347 potential HRD1-interacting proteins in HepG2 liver cells. Statistical filtering using COMPASS and SAINT computational scoring yielded 75 high-confidence interaction partners (p < 0.01) .

• Validation strategy: After identifying potential interactions, confirm them in both overexpression systems (e.g., transiently transfected HEK293 cells) and endogenous conditions using reciprocal Co-IP experiments .

• Functional analysis: Examine the effect of HRD1 on substrate protein levels through genetic manipulation (knockout/knockdown) followed by immunoblotting, as shown in HRD1-deficient liver tissues where ENTPD5, HSD17B4, CPT2, and RMND1 protein levels increased 2-5 fold without corresponding mRNA changes .

What approaches can effectively assess HRD1's role in T cell activation and function?

To investigate HRD1's role in T cell activation:

• Knockout model generation: Develop T cell-specific HRD1 knockout models to study the direct impact of HRD1 deficiency on T cell functions .

• Proliferation assessment: Use standard proliferation assays (e.g., CFSE dilution, BrdU incorporation) to evaluate T cell expansion. HRD1 deficiency has been shown to inhibit T cell proliferation .

• Cell cycle analysis: Examine cell cycle progression, particularly focusing on G1/S transition, as HRD1 targets p27^Kip1 (a cyclin-dependent kinase inhibitor) for ubiquitination and degradation .

• T cell differentiation: Analyze T helper cell subtype differentiation (Th1, Th17) through cytokine production profiling and transcription factor analysis. HRD1 deletion inhibits differentiation of both Th1 and Th17 cells .

• Mechanistic investigation: Examine the ubiquitination status and expression levels of key T cell signaling regulators, particularly p27^Kip1, in the presence and absence of HRD1 .

• Disease models: For functional relevance, test the impact of HRD1 manipulation in autoimmune disease models, such as experimental autoimmune encephalomyelitis (EAE) .

How do I design experiments to distinguish between ERAD-dependent and ERAD-independent functions of HRD1?

HRD1 exhibits both ERAD-dependent and ERAD-independent functions. To distinguish between these:

• Domain-specific mutations: Generate HRD1 constructs with mutations in specific domains (RING domain for ubiquitin ligase activity, transmembrane domain for ER localization) and assess their ability to rescue phenotypes in HRD1-deficient cells.

• Substrate localization analysis: Determine cellular localization of putative HRD1 substrates. ERAD substrates typically localize to the ER, while ERAD-independent substrates may be cytosolic or nuclear.

• ERAD blockade: Use chemical inhibitors of the ERAD pathway (e.g., proteasome inhibitors) or genetic manipulation of other ERAD components, and assess whether HRD1-mediated phenotypes persist.

• Comparative analysis: Compare HRD1's regulation of MHC-I (ERAD-dependent through misfolded β2-microglobulin degradation) versus MHC-II (ERAD-independent through Blimp1 regulation) .

• Organelle fractionation: Isolate different cellular compartments to determine where HRD1-substrate interactions occur, distinguishing between ER-localized (likely ERAD-dependent) and non-ER (likely ERAD-independent) interactions .

What controls should be included when using HRD1 antibodies for immunological research?

When using HRD1 antibodies in immunological research, include these essential controls:

• Positive tissue/cell controls:

  • Ramos cells (B cell line)

  • Activated T cells (HRD1 is induced by TCR/CD28 signaling)

  • Dendritic cells (for antigen presentation studies)

• Negative controls:

  • HRD1 knockout/knockdown cells or tissues

  • Isotype control antibodies for immunoprecipitation (e.g., normal rabbit IgG)

  • Secondary antibody-only controls for immunofluorescence

• Specificity validation:

  • Peptide competition assays

  • Multiple antibodies targeting different epitopes of HRD1

  • siRNA knockdown to confirm signal reduction

• Functional validation:

  • For studies on T cell function, include analysis of known HRD1 substrates (e.g., p27^Kip1)

  • For antigen presentation studies, include MHC-I and MHC-II expression analysis

These controls ensure the specificity of observed results and help distinguish between direct and indirect effects of HRD1 in immunological processes.

How do I optimize immunoprecipitation conditions for studying HRD1 interactions?

For successful immunoprecipitation of HRD1 and its interaction partners:

• Lysis buffer optimization:

  • Use a mild buffer (e.g., 1% NP-40 or CHAPS) for membrane protein extraction

  • Include protease inhibitors to prevent degradation

  • Add deubiquitinase inhibitors (e.g., N-ethylmaleimide) to preserve ubiquitination status

  • Consider including mild detergents that preserve membrane protein interactions

• Antibody selection and concentration:

  • Use 1-5 μg of HRD1 antibody per sample

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Include IgG control for comparison

• Cross-linking considerations:

  • For transient interactions, consider using reversible cross-linking agents

  • For ubiquitination studies, treat cells with proteasome inhibitors (e.g., MG132) before lysis

• Validation approach:

  • Perform reciprocal Co-IPs (immunoprecipitate with antibodies against interaction partners)

  • Confirm interactions in both overexpression systems and endogenous conditions

  • Validate using multiple cell types (e.g., HEK293, HepG2)

• Detection method:

  • Use ECL for standard detection

  • Consider more sensitive methods for detecting low-abundance interaction partners

What are the key considerations for analyzing HRD1 in liver metabolism studies?

When studying HRD1's role in liver metabolism:

• Model systems:

  • Use liver-specific HRD1 knockout mice (e.g., HRD1^Alb mice) to study in vivo functions

  • HepG2 cells provide a reliable in vitro model for studying HRD1 interactions

• Metabolic challenge:

  • Challenge mice with high-fat diet (HFD) to reveal HRD1's role in obesity and lipid metabolism

  • Analyze both fasting and refeeding conditions to capture dynamic metabolic responses

• Comprehensive analyses:

  • Measure body weight, serum glucose, triglycerides, and cholesterol

  • Analyze liver lipid content through histological and biochemical approaches

  • Combine with transcriptomic analysis to identify affected metabolic pathways

• Target substrate analysis:

  • Focus on HRD1 metabolic interactors: ENTPD5, CPT2, RMND1, HSD17B4, and ATP5D

  • Analyze both protein and mRNA levels to distinguish transcriptional from post-translational regulation

• Signaling pathway assessment:

  • Examine AMPK and AKT pathway activation status

  • Analyze expression of lipogenic genes like SREBP1 and SCD1

How do I troubleshoot inconsistent HRD1 detection in western blotting?

When encountering inconsistent HRD1 detection:

• Sample preparation issues:

  • Ensure complete solubilization of membrane proteins (HRD1 is an ER membrane protein)

  • Add fresh protease inhibitors to prevent degradation

  • Avoid repeated freeze-thaw cycles of samples

• Antibody-specific considerations:

  • Verify antibody specificity using positive controls (HepG2, 293T, HeLa cells)

  • Test different antibody lots or sources if inconsistency persists

  • Optimize antibody concentration (starting from 1:1000 dilution)

• Technical adjustments:

  • Increase protein loading (10-20 μg may be insufficient for tissues with low HRD1 expression)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use more sensitive detection methods (ECL-plus or fluorescent secondaries)

  • Optimize transfer conditions for high molecular weight proteins

• HRD1 expression variability:

  • Consider that HRD1 expression is induced postprandially in liver tissues

  • Check ER stress conditions, as this may affect HRD1 levels

  • Account for TCR/CD28 stimulation effects when studying T cells

How do I interpret conflicting data on HRD1 function across different tissues?

HRD1 exhibits tissue-specific functions, which can lead to apparently conflicting data. When interpreting such results:

• Tissue-specific substrate profiles:

  • HRD1 targets different substrates in different tissues (e.g., metabolic enzymes in liver, p27^Kip1 in T cells)

  • Perform tissue-specific interactome studies to identify relevant substrates

• Context-dependent signaling:

  • In liver, HRD1 deletion activates both AMPK and AKT pathways

  • In T cells, HRD1 promotes TCR/CD28 signaling

  • Analyze pathway-specific markers in each tissue context

• Phenotypic differences:

  • Liver-specific deletion is protective against metabolic disorders

  • T cell-specific deletion is protective against autoimmunity

  • These seemingly opposing effects reflect tissue-specific roles

• Experimental design considerations:

  • Use tissue-specific conditional knockout models rather than global knockouts

  • Combine in vitro and in vivo approaches to validate findings

  • Consider developmental versus acute roles through inducible deletion systems

• Mechanistic reconciliation:

  • HRD1 operates through both ERAD-dependent and independent mechanisms

  • The balance between these functions may differ by tissue or condition

How do I effectively study the dual role of HRD1 in both protein quality control and signaling regulation?

To study HRD1's dual functionality:

• Substrate differentiation:

  • Classify substrates as misfolded proteins (quality control) or functional proteins (signaling)

  • Determine substrate half-life and ubiquitination patterns (K48 vs. K63 linkages)

  • Assess whether substrate regulation is stress-dependent or constitutive

• Compartmentalization analysis:

  • Use subcellular fractionation and immunofluorescence to determine where HRD1-substrate interactions occur

  • Compare ER-localized interactions with cytosolic interactions

• Structure-function analysis:

  • Generate HRD1 mutants lacking specific domains or functions

  • Test these mutants for complementation of different HRD1-dependent phenotypes

• Temporal dynamics:

  • Use inducible systems to distinguish between acute and chronic effects of HRD1 manipulation

  • Analyze both basal and stress-induced conditions

• Integrated approach:

  • Combine proteomic (for substrates), transcriptomic (for downstream effects), and metabolomic (for functional outcomes) analyses

  • Use computational modeling to integrate these multi-omics datasets