HERPUD1 Antibody

What is HERPUD1 and what cellular functions does it serve?

HERPUD1 is a component of the endoplasmic reticulum quality control (ERQC) system, also called ER-associated degradation (ERAD), which is involved in ubiquitin-dependent degradation of misfolded endoplasmic reticulum proteins . The protein has a structure that protrudes from the ER membrane into the cytosol, with a ubiquitin-like (UBL) domain at its N-terminus . HERPUD1 expression is strongly upregulated by ER stress and was first identified as an ER stress-responsive protein in human vascular endothelial cells .

Functionally, HERPUD1 contributes to the degradation of misfolded proteins through the ERAD pathway. It binds to ubiquilins, and this interaction is required for efficient degradation of CD3D via the ERAD pathway . Additionally, research indicates that HERPUD1 may enhance presenilin-mediated amyloid-beta protein 40 generation, suggesting potential roles in neurodegenerative disease mechanisms .

What applications are HERPUD1 antibodies validated for?

HERPUD1 antibodies have been validated for multiple experimental applications:

The antibodies have been tested for reactivity with human samples primarily, though some cross-reactivity with mouse samples has been reported .

What are the optimal storage and handling conditions for HERPUD1 antibodies?

For optimal antibody performance, HERPUD1 antibodies should be stored at -20°C, where they remain stable for one year after shipment . Most commercial preparations are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage for smaller (20μl) sizes that contain 0.1% BSA .

When performing antigen retrieval for immunohistochemistry applications, it is recommended to use TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative . Each antibody should be titrated in specific testing systems to achieve optimal results, as optimal concentration may be sample-dependent .

What explains the discrepancy between calculated (44 kDa) and observed (54 kDa) molecular weight for HERPUD1?

The calculated molecular weight of HERPUD1 is approximately 44 kDa, but the observed molecular weight in Western blot applications is often around 54 kDa . This discrepancy is likely due to post-translational modifications, particularly phosphorylation and glycosylation, which can alter protein mobility during SDS-PAGE.

When performing Western blot analysis of HERPUD1, researchers should anticipate this size difference and optimize separation conditions accordingly. Using gradient gels (7.5-10%) may provide better resolution of HERPUD1, as demonstrated in validation studies where both 7.5% and 10% SDS-PAGE systems were used successfully . When validating antibody specificity, knockout/knockdown controls are essential to confirm band identity.

How can researchers effectively detect and quantify ER stress-induced HERPUD1 upregulation?

Since HERPUD1 is strongly upregulated during ER stress, experimental designs should include appropriate stress induction models. Based on validation data, tunicamycin-treated HepG2 cells show increased HERPUD1 expression and can serve as a positive control for antibody validation .

For quantitative analysis:

• Include time-course experiments (0-48 hours) after ER stress induction to capture the dynamic expression profile of HERPUD1

• Use multiple ER stress inducers (tunicamycin, thapsigargin, DTT) to distinguish pathway-specific responses

• Perform parallel analysis of other ER stress markers (BiP/GRP78, CHOP, XBP1 splicing) to contextualize HERPUD1 regulation

• Normalize HERPUD1 protein levels to appropriate housekeeping proteins that remain stable during ER stress

Western blot analysis should be complemented with qRT-PCR to distinguish between transcriptional upregulation and post-translational stabilization of HERPUD1 during the stress response.

How can HERPUD1 antibodies be used to investigate mechanisms of cardiac hypertrophy?

Research has revealed that HERPUD1 may function as an endogenous suppressor of cardiac hypertrophy . When designing experiments to investigate this role, researchers should consider:

• Cell models: H9C2 cells (rat cardiomyocytes) are an established model system for studying HERPUD1's role in cardiac hypertrophy .

• Hypertrophy induction: Angiotensin II (Ang II) at 1μM concentration can be used to induce hypertrophy in vitro, mimicking pathological stress .

• Manipulation approaches:

  • For loss-of-function: Transfect cells with 25nM HERPUD1 siRNA using appropriate transfection reagents (e.g., TransIT-TKO)

  • For gain-of-function: Transfect 2ng/μL of HERPUD1-EGFP plasmid

• Analysis timepoints: Evaluate hypertrophic responses 48 hours post-transfection or treatment .

• Readouts: Assess cell size through immunofluorescence staining and quantitative image analysis; monitor nuclear translocation of NFATc4 (a hypertrophy-related transcription factor) through subcellular fractionation and immunoblotting .

The experimental data suggests that silencing HERPUD1 increases cell size approximately 1.55-fold compared to controls, while overexpression of HERPUD1 prevents Ang II-induced hypertrophy . When analyzing NFATc4 translocation, both cytosolic and nuclear fractions should be examined, as HERPUD1 inhibits the nuclear translocation of this transcription factor .

What techniques are recommended for studying HERPUD1's interactions with calcium-handling proteins?

HERPUD1 interacts with calcium-release proteins such as IP3R and RyR, potentially regulating their degradation and function . When investigating these interactions:

• Protein-protein interaction studies:

  • Co-immunoprecipitation using HERPUD1 antibodies to pull down associated calcium channels

  • Proximity ligation assays to visualize interactions in situ

  • FRET-based approaches for live-cell interaction dynamics

• Degradation analysis:

  • Cycloheximide chase assays to compare IP3R/RyR turnover rates in the presence or absence of HERPUD1

  • Ubiquitination assays to assess HERPUD1's impact on calcium channel ubiquitination

• Functional measurements:

  • Calcium imaging using fluorescent indicators to assess cytosolic Ca2+ levels

  • Patch-clamp electrophysiology to measure channel activity

  • ER Ca2+ measurements using targeted sensors

Research has shown that HERPUD1 knockout or knockdown leads to increased IP3R protein levels without affecting mRNA levels, suggesting post-translational regulation . When designing experiments, consider that HERPUD1-mediated effects may be particularly pronounced under ER stress conditions, as HERPUD1 expression is stress-inducible.

What controls should be included when using HERPUD1 antibodies in Western blot applications?

Rigorous experimental design for HERPUD1 detection by Western blot should include:

• Positive controls:

  • Tunicamycin-treated HepG2 cells (confirmed to express HERPUD1)

  • BxPC-3, HeLa, LNCaP, or U2OS cell lysates (validated positive expression)

  • Mouse brain whole cell lysate (for cross-reactivity validation)

• Negative controls:

  • HERPUD1 knockdown/knockout samples using siRNA or CRISPR-Cas9

  • Non-relevant cell types with minimal HERPUD1 expression

  • Secondary antibody-only controls to assess non-specific binding

• Loading controls:

  • Standard housekeeping proteins (β-actin, GAPDH, α-tubulin)

  • Total protein staining methods (Ponceau S, REVERT)

• Molecular weight markers:

  • Include precision markers that span 40-60 kDa range to accurately assess the observed 54 kDa band

• Antibody concentration optimization:

  • Test different dilutions (1:500, 1:1000, 1:5000) as optimal concentration may vary by sample type

The expected band size is approximately 54 kDa despite the calculated molecular weight of 44 kDa . Using gradient gels or different percentage SDS-PAGE (7.5-10%) may help to optimize band resolution and separation .

How can subcellular localization of HERPUD1 be accurately determined using immunofluorescence?

HERPUD1 is primarily localized to the endoplasmic reticulum membrane, with portions extending into the cytosol . For accurate subcellular localization:

• Co-staining approach:

  • ER markers: Co-stain with established ER markers (calnexin, calreticulin, PDI)

  • Nuclear counterstain: DAPI or Hoechst for nuclear visualization

  • Other organelles: Mitochondria (MitoTracker), Golgi (GM130) for exclusion verification

• Fixation and permeabilization optimization:

  • Test both paraformaldehyde (4%) and methanol fixation methods

  • Compare different permeabilization agents (0.1-0.5% Triton X-100, 0.05% saponin)

  • Evaluate antigen retrieval impact on signal intensity and specificity

• Antibody validation:

  • Include HERPUD1-GFP fusion protein as a reference for localization pattern

  • Perform parallel knockdown experiments to confirm specificity of staining

  • Use both monoclonal and polyclonal antibodies when possible to verify patterns

• Advanced imaging:

  • Super-resolution microscopy (STED, STORM) for detailed localization

  • Confocal z-stacking for three-dimensional localization analysis

  • Live-cell imaging using fluorescently tagged HERPUD1 to monitor dynamic localization

When analyzing HERPUD1 localization during stress responses, time-course experiments should be performed to capture potential translocation events or changes in distribution patterns.

How can HERPUD1 antibodies be utilized to study its role in cardiac pathology?

HERPUD1 appears to have a protective role against cardiac hypertrophy, with evidence showing that Herpud1 knockout mice develop cardiac hypertrophy characterized by increased heart weight and left ventricular wall thickness . When investigating HERPUD1's role in cardiac pathology:

• Animal models:

  • Transverse aortic coarctation (TAC) models with wild-type and Herpud1 knockout mice

  • Angiotensin II infusion models

  • Heart failure models to assess HERPUD1 expression changes

• Tissue analysis approaches:

  • Immunohistochemistry of cardiac sections to assess HERPUD1 expression patterns

  • Western blot analysis of heart lysates from different cardiac regions

  • RNA analysis (qPCR, RNA-seq) to correlate protein with transcript levels

• Functional correlations:

  • Echocardiography parameters correlated with HERPUD1 expression

  • Cardiomyocyte size measurements in relation to HERPUD1 levels

  • Calcium handling parameters (transients, spark frequency, wave propagation)

• Intervention studies:

  • Viral vector-mediated overexpression of HERPUD1 in hypertrophic models

  • Pharmacological induction of HERPUD1 as a potential therapeutic approach

Studies have shown that overexpression of HERPUD1 suppresses Ang II-induced cell hypertrophy and inhibits nuclear translocation of NFATc4, suggesting that HERPUD1 might function as an anti-hypertrophic gene . These findings indicate that modulation of HERPUD1 expression or activity could have therapeutic potential in cardiac hypertrophy management.

What strategies can be employed to investigate HERPUD1's role in the ERAD pathway?

As a component of the endoplasmic reticulum quality control system, HERPUD1 participates in the degradation of misfolded proteins through the ERAD pathway . To investigate this role:

• Substrate degradation assays:

  • Use model ERAD substrates (CD3δ, TCRα, mutant CFTR)

  • Monitor substrate half-life in the presence or absence of HERPUD1

  • Employ cycloheximide chase experiments with quantitative Western blotting

• Ubiquitination analysis:

  • Immunoprecipitate ERAD substrates and blot for ubiquitin

  • Use K48-specific ubiquitin antibodies to focus on degradation-targeted substrates

  • Perform in vitro ubiquitination assays with recombinant components

• Protein-protein interaction mapping:

  • Identify HERPUD1 interactors using co-immunoprecipitation followed by mass spectrometry

  • Map interactions with known ERAD components (HRD1, SEL1L, p97/VCP)

  • Perform domain mapping to identify interaction interfaces

• Stress induction experiments:

  • Compare ERAD efficiency under normal and ER stress conditions

  • Induce misfolded protein accumulation with tunicamycin, thapsigargin, or DTT

  • Assess HERPUD1's contribution to cell survival during prolonged ER stress

Research has demonstrated that HERPUD1 interacts with ubiquilins, and this interaction is required for efficient degradation of CD3D via the ERAD pathway . Additionally, HERPUD1 has been shown to interact with calcium-release proteins such as IP3R and RyR, potentially regulating their degradation . These findings suggest that HERPUD1 may serve as a selective adaptor in the ERAD pathway, targeting specific substrates for degradation.

How can researchers investigate the relationship between HERPUD1 and calcium signaling pathways?

HERPUD1 has been linked to calcium homeostasis through its interactions with IP3Rs and RyRs . To explore this relationship:

• Calcium imaging experiments:

  • Compare baseline and stimulated calcium responses in HERPUD1 knockdown/overexpression models

  • Use targeted calcium indicators to measure ER, mitochondrial, and cytosolic calcium pools

  • Perform calcium oscillation analysis to assess signaling dynamics

• Channel regulation studies:

  • Measure IP3R and RyR protein levels in HERPUD1-manipulated cells

  • Determine if HERPUD1 affects channel open probability using single-channel recordings

  • Investigate if HERPUD1 modulates channel phosphorylation or other post-translational modifications

• Pathophysiological relevance:

  • Examine if calcium dysregulation in HERPUD1-deficient systems contributes to disease phenotypes

  • Test if restoring calcium homeostasis rescues HERPUD1 knockout phenotypes

  • Investigate potential compensatory mechanisms that emerge with chronic HERPUD1 deficiency

Studies have shown that Herpud1 knockout mice and siHerpud1-treated NRVMs exhibit increased IP3R protein levels without changes in mRNA levels, suggesting post-translational regulation . This leads to increased cytosolic Ca2+ levels, which may contribute to hypertrophic signaling in cardiomyocytes .

What approaches can be used to study the therapeutic potential of targeting HERPUD1 in cardiac disease?

Given HERPUD1's protective role against cardiac hypertrophy , exploring its therapeutic potential requires:

• Pharmacological modulation:

  • Screen for compounds that induce HERPUD1 expression

  • Test ER stress modulators for their impact on HERPUD1 levels and cardiac hypertrophy

  • Develop targeted approaches to stabilize HERPUD1 protein or enhance its activity

• Gene therapy approaches:

  • Design cardiac-specific HERPUD1 overexpression vectors

  • Optimize delivery methods for cardiomyocyte targeting

  • Evaluate long-term effects of HERPUD1 overexpression on cardiac function

• Biomarker development:

  • Assess if circulating HERPUD1 levels correlate with cardiac disease states

  • Develop methods to measure HERPUD1 activity or modifications as potential biomarkers

  • Evaluate if HERPUD1 genetic variants associate with cardiac disease susceptibility

• Combination therapies:

  • Test HERPUD1-targeting approaches alongside standard heart failure medications

  • Investigate synergies with calcium channel blockers or NFAT inhibitors

  • Explore complementary approaches targeting other ERAD components

Research has demonstrated that overexpression of HERPUD1 suppresses Ang II-induced cardiac hypertrophy by inhibiting the nuclear translocation of NFATc4 . This mechanism suggests that enhancing HERPUD1 expression or activity could represent a novel therapeutic strategy for preventing or treating pathological cardiac hypertrophy.