HIP1R Antibody

What is HIP1R and what cellular functions does it regulate?

HIP1R (Huntingtin-interacting protein 1-related protein) is a 138 kDa protein involved in several critical neuronal functions. It plays a significant role in regulating synaptic transmission, particularly affecting the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Knockdown studies have demonstrated that HIP1R reduction specifically impacts mEPSCs without affecting miniature inhibitory postsynaptic currents . Additionally, HIP1R has been identified as a protein capable of targeting histone deacetylase-3-mediated neurodegeneration, functioning alongside other proteins such as NPTX1, NFL, TEX10, and TGFFG . More recent research has revealed HIP1R's important role in regulating programmed death-ligand 1 (PD-L1), suggesting its involvement in immune checkpoint regulation .

What are the recommended applications and dilutions for HIP1R antibodies?

Based on validated research protocols, HIP1R antibodies can be effectively used in several common laboratory applications with specific recommended dilutions:

The antibody shows cross-reactivity with human, mouse, and rat samples, making it versatile for comparative studies across these species . When preparing for your experiments, avoid repeated freeze-thaw cycles as manufacturers typically recommend not to aliquot the antibody to maintain its stability and activity .

How should immunofluorescence microscopy be performed using HIP1R antibodies?

For effective immunofluorescence microscopy with HIP1R antibodies, follow this validated protocol:

• Culture cells on coverslips in appropriate growth medium

• Transfect cells with HIP1R-tagged constructs if studying exogenous protein

• For stimulation experiments, treat cells with the appropriate ligand (e.g., EGF at 1 μg/ml) at 4°C for 30 minutes

• Change medium to 37°C for the desired time intervals to observe dynamic changes

• Fix cells in 4% paraformaldehyde at 4°C for 20 minutes

• Block with appropriate blocking solution

• Incubate with primary HIP1R antibody (typically at 1:100 dilution)

• Apply fluorophore-conjugated secondary antibodies

• Image using confocal microscopy with 63× water immersion objective

• For colocalization analysis, use software like ImageJ with JACoP plug-in to calculate Manders coefficients with automated thresholding

This protocol has been successfully used to visualize HIP1R localization and dynamics in response to receptor stimulation.

How can HIP1R expression be accurately quantified in tissue samples using immunohistochemistry?

For rigorous quantification of HIP1R expression in tissue samples, implement the following validated IHC protocol and scoring system:

• Perform immunohistochemical staining using an automated staining platform (such as Benchmark XT, Ventana) with anti-HIP1R antibody at 1:1000 dilution (16814-1-AP, polyclonal, Proteintech)

• Include appropriate positive controls (human placenta tissue is recommended)

• Evaluate staining intensity using a four-point scale:

  • 0: No staining

  • 1: Light yellow (faint staining)

  • 2: Yellow-brown (moderate staining)

  • 3: Brown (strong staining)

• Assess both cytoplasmic and membranous localization of HIP1R, recording percentages (0-100%)

• Calculate H-scores using the formula: H-score = [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]

• H-scores range from 0-300, providing a comprehensive quantitative measure of protein expression

This methodology has been successfully employed in clinical studies analyzing HIP1R expression in non-small cell lung cancer samples, with a cutoff H-score value of 180 (66% sensitivity and 68% specificity) identified as optimal for distinguishing between high and low expression groups .

What is the relationship between HIP1R expression and PD-L1 regulation, and how can researchers investigate this interaction?

The relationship between HIP1R and PD-L1 is complex and involves multiple immune-related pathways. Research has revealed:

• At protein level (IHC): No statistically significant correlation has been observed between HIP1R and PD-L1 expression in direct immunohistochemical analyses (p = 0.905)

• At mRNA level: HIP1R mRNA expression demonstrates significant negative correlation with PD-L1 mRNA levels in both:

  • Lung adenocarcinoma (Spearman's rho = −0.233, p < 0.001)

  • Squamous cell carcinoma (Spearman's rho = −0.224, p < 0.001)

• Functional significance: Low HIP1R expression predicts better response to anti-PD-1 therapy in NSCLC patients:

  • Univariate analysis: OR = 0.235, p = 0.015

  • Multivariate analysis: OR = 0.209, p = 0.014

To investigate this interaction, researchers should employ a multi-modal approach:

• Perform both protein (IHC) and mRNA expression analyses

• Conduct gene set enrichment analysis (GSEA) to identify associated pathways

• Focus particularly on allograft rejection, inflammatory responses, IL6-JAK-STAT3, IL2-STAT5, and interferon gamma response pathways, which have shown significant association with HIP1R expression

How can mass spectrometry be optimized for identification of HIP1R and its interacting proteins?

To optimize mass spectrometry analysis of HIP1R and its interacting proteins, implement this validated protocol:

• Scale preparation: Begin with substantial starting material (e.g., 100 10-cm dishes of cells providing approximately 100 mg protein)

• Stimulation: If studying dynamic interactions, stimulate cells with appropriate ligands (e.g., EGF for receptor tyrosine kinase pathways)

• Immunoprecipitation: Use validated anti-HIP1 antibodies (such as UM410) for efficient pull-down

• Gel separation: Perform 6% PAGE to isolate the 120-kDa HIP1R band

• In-gel digestion: Conduct tryptic digestion directly from the excised gel band

• Mass analysis: Perform MALDI-TOF analysis of digested peptides

• Database comparison: Use Mascot server (Matrix Sciences) to compare spectra against databases such as Swiss-Prot

• Verification: Confirm interactions using complementary approaches such as co-immunoprecipitation or proximity ligation assays

This approach has successfully identified HIP1R tryptic polypeptides and associated proteins, providing insights into its interaction network and post-translational modifications.

How does HIP1R expression correlate with clinical outcomes in cancer patients?

HIP1R expression has demonstrated significant prognostic value in cancer patients, particularly those receiving immunotherapy:

These findings suggest that HIP1R antibody-based assays may have clinical utility in predicting immunotherapy outcomes. For researchers designing predictive biomarker studies, incorporating HIP1R expression analysis alongside established markers like PD-L1 may improve patient stratification accuracy.

What are the optimal approaches for multiplexed analysis of HIP1R with other immune checkpoints?

For comprehensive investigation of HIP1R in relation to other immune checkpoints, researchers should implement these validated approaches:

• Multiplex immunohistochemistry:

  • Use sequential IHC staining with HIP1R antibody (16814-1-AP, 1:1000) and antibodies against PD-L1, PD-1, and other immune checkpoint molecules

  • Employ multispectral imaging systems to differentiate multiple antibody signals

  • Analyze spatial relationships between HIP1R and immune checkpoint molecules in the tumor microenvironment

• Transcriptomic analysis:

  • Perform RNA-seq or targeted NanoString analysis to simultaneously quantify HIP1R and multiple immune-related genes

  • Conduct gene set enrichment analysis (GSEA) focusing specifically on HIP1R-associated immune pathways:

    • HALLMARK_ALLOGRAFT_REJECTION

    • HALLMARK_INFLAMMATORY_RESPONSE

    • HALLMARK_IL6_JAK_STAT3_SIGNALING

    • HALLMARK_IL2_STAT5_SIGNALING

    • HALLMARK_INTERFERON_GAMMA_RESPONSE

• Integration of protein and mRNA data:

  • Address the observed discrepancy between protein-level and mRNA-level correlations between HIP1R and PD-L1

  • Investigate post-transcriptional regulatory mechanisms that may mediate HIP1R's effect on PD-L1

This multi-modal approach will provide comprehensive insights into HIP1R's role within the complex network of immune checkpoint regulation.

What are common pitfalls in Western blotting with HIP1R antibodies and how can they be resolved?

When performing Western blotting with HIP1R antibodies, researchers frequently encounter several challenges. Here are the most common issues and their solutions:

• Detection of high molecular weight protein (138 kDa):

  • Use lower percentage gels (6-8%) to ensure adequate separation

  • Extend transfer time or use specialized transfer systems for large proteins

  • Verify complete transfer using reversible staining methods before antibody incubation

• Non-specific bands:

  • Optimize blocking conditions (5% non-fat milk or 5% BSA in TBS-T)

  • Use highly purified primary antibody at the recommended 1:1000 dilution

  • Include appropriate controls: positive control (tissue known to express HIP1R), negative control (knockout or knockdown samples)

• Weak signal:

  • Increase protein loading (start with 50-75 μg total protein)

  • Ensure protein isn't degraded by using fresh lysates with appropriate protease inhibitors

  • Optimize primary antibody incubation (overnight at 4°C)

  • Use enhanced chemiluminescence (ECL) detection systems with extended exposure times

  • Consider HRP-conjugated secondary antibodies at 1:5,000 dilution

• Signal variability between experiments:

  • Standardize lysate preparation and storage conditions

  • Include loading controls appropriate for your experimental conditions

  • Consider using the same positive control sample across multiple blots for normalization

How can researchers optimize immunoprecipitation protocols for studying HIP1R interactions?

For successful immunoprecipitation of HIP1R and identification of its interacting partners, implement these optimized protocol elements:

• Antibody selection and concentration:

  • Use antibodies specifically validated for immunoprecipitation

  • Apply at 1:100 dilution as recommended for optimal capture efficiency

  • Consider using tag-specific antibodies (e.g., anti-Myc) for tagged constructs to increase specificity

• Lysis conditions:

  • Use mild lysis buffers to preserve protein-protein interactions

  • Include phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Maintain cold temperatures throughout to prevent complex dissociation

• Pre-clearing strategy:

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

  • Use species-matched IgG controls to identify non-specific interactions

• Confirmation approaches:

  • Verify interactions by reciprocal immunoprecipitation

  • Combine with proximity ligation assays in intact cells

  • For transient interactions, consider crosslinking approaches prior to lysis

• Scale considerations:

  • For mass spectrometry analysis, scale up to approximately 100 mg starting protein

  • For targeted co-IP Western blot analysis, 1-2 mg total protein is typically sufficient

Following these optimized protocols will significantly improve the specificity and yield of HIP1R immunoprecipitation experiments, facilitating the discovery of physiologically relevant interaction partners.