GSTA4 Antibody

What are the optimal protocols for GSTA4 immunohistochemistry in tissue sections?

GSTA4 immunohistochemistry requires specific preparation techniques to maintain antibody sensitivity while preserving tissue architecture. Based on published methodologies:

Recommended protocol:

• Fix tissue samples in formalin and embed in paraffin

• Deparaffinize and hydrate tissues using standard procedures

• Block endogenous peroxidase activity with 3% hydrogen peroxide (10 minutes)

• Perform antigen retrieval by microwaving for 15 minutes in 10 mM citrate buffer (pH 6.0)

• Allow cooling for 20 minutes

• Block non-specific binding with appropriate blocking reagent (e.g., Background Sniper)

• Incubate with anti-GSTA4 antibody (1:5000 dilution has been validated)

• Apply appropriate secondary antibody (e.g., Envision plus anti-rabbit with HRP-labeled polymer)

• Develop using diaminobenzidine as chromogen

• Counterstain as needed for cell identification

This protocol has been successfully used to detect GSTA4 in multiple mouse tissues, including epidermis, stria vascularis, spiral ganglion neurons, and organ of Corti .

What are the recommended dilutions for different GSTA4 antibody applications?

The optimal dilutions vary based on application and specific antibody. A comprehensive dilution guide for typical research applications:

These ranges are based on validated commercial antibodies, but optimization for your specific experimental system is essential .

How can I validate the specificity of my GSTA4 antibody?

Antibody specificity validation is critical for reliable results. Multiple complementary approaches are recommended:

• Genetic knockout controls: Compare staining between wild-type and Gsta4-/- tissues. Complete absence of signal in knockout tissue confirms specificity .

• Western blot verification: Confirm the antibody detects a single band at the expected molecular weight (approximately 27 kDa for GSTA4) .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before staining to block specific binding.

• Cross-species reactivity testing: If working across species, verify antibody reactivity in your model organism.

• Multiple detection methods: Confirm GSTA4 expression using complementary techniques (e.g., qPCR validation of protein expression patterns) .

The most rigorous validation uses Gsta4-deficient tissues, which should show complete absence of immunostaining when using a specific GSTA4 antibody .

What is the optimal protocol for Western blot detection of GSTA4?

For robust Western blot detection of GSTA4:

• Sample preparation:

  • Lyse cells/tissues in RIPA buffer containing protease and phosphatase inhibitors

  • Load 30 μg protein per lane on 10% SDS-PAGE gels

• Transfer and blocking:

  • Transfer to PVDF membrane

  • Block with 5% bovine serum albumin in TBST

• Antibody incubation:

  • Incubate with anti-GSTA4 antibody (1:1000-1:7500 dilution range has been validated)

  • Use β-actin (1:10,000) as loading control

  • Incubate overnight at 4°C

• Detection:

  • Wash 3× with TBST (10 minutes each)

  • Incubate with HRP-conjugated secondary antibody

  • Wash 4× with TBST (15 minutes each)

  • Visualize using chemiluminescence detection

GSTA4 consistently appears as a 27 kDa band on Western blots .

What are the best cell fixation methods for GSTA4 immunofluorescence?

For optimal immunofluorescence detection of GSTA4:

• Fixation options:

  • Pre-cold acetone fixation has been successfully used for GSTA4 immunostaining

  • Alternative: 4% paraformaldehyde (10 minutes at room temperature)

• Permeabilization:

  • 0.1% Triton X-100 for cell permeabilization

  • Block with 1% BSA in PBS

• Antibody incubation:

  • Primary: Incubate with anti-GSTA4 antibody (1:50-1:500 dilution) overnight at 4°C

  • Secondary: FITC-conjugated anti-rabbit secondary antibody (typically 1:60-1:200)

  • Counterstain nuclei with DAPI

• Visualization:

  • Examine using confocal or fluorescence microscopy (200× magnification has been used successfully)

This approach allows visualization of cellular and subcellular GSTA4 distribution patterns.

How can I design experiments to investigate GSTA4's role in cancer cell proliferation and tumorigenesis?

GSTA4 has demonstrated roles in both promoting and suppressing tumor growth, depending on cancer type. To investigate these functions:

Recommended experimental design:

• Genetic manipulation approaches:

  • Gene knockout: Use CRISPR-Cas9 to generate GSTA4-deficient cancer cell lines

    • Design guide RNAs targeting exonic regions (e.g., gRNA66 and gRNA214 have been used)

    • Transfect using Lipofectamine 3000

    • Screen with puromycin (4 μg/ml initial, 1 μg/ml maintenance)

    • Verify deletion by PCR and sequencing

  • Knockdown: Use GSTA4-specific siRNA (e.g., Santa Cruz Biotechnology, sc-105424)

  • Overexpression: Generate stable overexpression lines using retroviral vectors (e.g., pQCXIP-GSTA4)

• Functional assays:

  • Proliferation: EdU incorporation assay or MTT/MTS assays at 24, 48, and 72h timepoints

  • Colony formation: Assess clonogenicity in semi-solid agar

  • Migration/invasion: Transwell invasion assay (1×10^5 cells in upper chamber, 10% FBS in lower chamber, 24h incubation)

• Pathway analysis:

  • Assess activation status of AKT, p38 MAPK, GSK-3β, mTOR, and FAK pathways via Western blot

  • Measure phosphorylation of key sites (AKT Ser473, GSK-3β Ser-9, mTOR Ser2448)

GSTA4 manipulation significantly impacts cancer cell behavior. For example, knockout of GSTA4 in HCT116 colorectal cancer cells decreased proliferation, clonogenicity, and EdU incorporation (39.1±4.6% vs. 32.6±2.9% EdU-positive cells, p<0.05) .

What are the optimal techniques for studying GSTA4's role in detoxification of 4-hydroxynonenal (4-HNE)?

4-HNE is a cytotoxic product of lipid peroxidation that GSTA4 efficiently detoxifies. To study this crucial function:

• 4-HNE detection methods:

  • Immunohistochemistry using anti-4-HNE antibodies to visualize 4-HNE-protein adducts

  • ELISA-based quantification of 4-HNE levels

  • Mass spectrometry to identify 4-HNE-modified proteins

• GSTA4 activity assays:

  • Spectrophotometric assays using 4-HNE as substrate

  • Measure GSH conjugation to 4-HNE in tissue/cell extracts

  • Compare activity between wild-type and GSTA4-deficient samples

• Oxidative stress models:

  • Chemical inducers: Cisplatin, paraquat, or H₂O₂ treatment

  • UV irradiation

  • Compare 4-HNE accumulation in wild-type vs. GSTA4-deficient models

• In vivo approaches:

  • Compare 4-HNE levels in tissues from wild-type vs. Gsta4-/- mice

  • Correlate with functional outcomes (e.g., tissue damage, apoptosis)

In cisplatin-induced ototoxicity studies, female Gsta4-/- mice showed significantly increased 4-HNE levels in cochlear neurons compared to male Gsta4-/- mice, correlating with greater hearing loss and neuronal damage .

How should I investigate tissue-specific and sex-dependent differences in GSTA4 expression and function?

GSTA4 exhibits significant tissue-specific and sex-dependent expression patterns that affect its function. To investigate these differences:

• Tissue distribution analysis:

  • Perform comprehensive immunohistochemical mapping across multiple tissue types

  • Compare expression patterns between normal and pathological states

  • Specific tissues of interest include:

    • Liver: Hepatocytes, bile duct cells, vascular endothelial cells

    • Skin: Upper layers of keratinocytes, sebaceous glands, sweat glands

    • Kidney: Proximal convoluted tubules

    • Colon: Epithelial cells of mucosa, muscle cells

    • Brain: Neurons

• Sex-dependent studies:

  • Compare GSTA4 mRNA and protein expression between males and females

  • Analyze enzyme activity levels by sex

  • Investigate hormonal influences through gonadectomy models

  • Design experiments with balanced sex representation

• Hormonal regulation:

  • Investigate estrogen's effect on GSTA4 expression using ovariectomy models

  • Measure Nrf2 expression (a regulator of Phase II enzymes including GSTA4)

  • Correlate with GSTA4 levels and activity

Research shows significant sex-dependent GSTA4 activity in response to stressors. For example, cisplatin stimulates GSTA4 activity in female but not male wild-type mice inner ears. In CBA/CaJ mice, ovariectomy decreased both Gsta4 mRNA expression and GSTA4 protein levels, suggesting estrogen-mediated regulation .

What experimental approaches best reveal GSTA4's role in chemoresistance mechanisms?

GSTA4 contributes to chemoresistance in several cancer types. To investigate this function:

• Cell viability assays with chemotherapeutic agents:

  • Compare sensitivity in GSTA4-manipulated cells (knockout, knockdown, overexpression)

  • Test multiple drug concentrations and timepoints

  • Assess with MTT/MTS, colony formation, or flow cytometry

• DNA damage assessment:

  • Measure γH2AX expression (marker of double-stranded DNA breaks)

  • Compare between GSTA4-expressing and GSTA4-deficient cells

  • Analyze in response to different chemotherapeutic agents (e.g., 5-FU, oxaliplatin, cisplatin)

• ROS measurement:

  • Quantify intracellular reactive oxygen species using fluorescent probes

  • Correlate with GSTA4 expression levels

  • Assess protective effect of GSTA4 against drug-induced oxidative stress

• In vivo xenograft studies:

  • Implant GSTA4-manipulated cancer cells in immunodeficient mice

  • Treat with chemotherapeutic agents

  • Measure tumor volume, growth rate, and response to therapy

Studies show GSTA4 knockout in HCT116 colorectal cancer cells increased sensitivity to 5-fluorouracil and oxaliplatin. 5-FU treatment resulted in higher γH2AX expression in GSTA4-deficient cells, indicating increased DNA damage. Similar results were observed in xenograft models, confirming GSTA4's role in chemoresistance both in vitro and in vivo .

How can I effectively study GSTA4's role in cellular signaling pathways?

GSTA4 affects multiple signaling pathways involved in proliferation, survival, and stress response. For comprehensive pathway analysis:

• Phosphorylation status assessment:

  • Examine key signaling nodes by Western blot:

    • AKT/mTOR pathway: phospho-AKT (Ser473), phospho-mTOR (Ser2448)

    • GSK-3β pathway: phospho-GSK-3β (Ser-9)

    • p38 MAPK pathway: phospho-p38

    • FAK/Src signaling: phospho-FAK (Tyr925), phospho-Src (Ser17)

• Pathway inhibitor studies:

  • Use specific inhibitors of identified pathways

  • Determine if inhibitors reverse GSTA4-dependent phenotypes

  • Identify critical pathways mediating GSTA4's effects

• Protein-protein interaction analysis:

  • Co-immunoprecipitation to identify binding partners

  • Proximity ligation assays for in situ interaction detection

  • Mass spectrometry to identify interaction networks

• Transcriptional responses:

  • RNA-seq to identify transcriptional changes

  • Focus on pathway-specific gene signatures

  • Compare wild-type vs. GSTA4-deficient cells

Research demonstrates GSTA4 inactivation in HCT116 cells blocks AKT and p38 MAPK pathways, contributing to proliferative suppression. These findings establish GSTA4 as a regulator of key signaling pathways involved in cancer cell proliferation and survival .

What are the most effective approaches for studying GSTA4 in neurodegenerative models?

GSTA4 plays critical roles in neuronal health and myelination. For neurodegenerative research:

• Oligodendrocyte differentiation models:

  • Study GSTA4's effect on oligodendrocyte precursor cell (OPC) differentiation

  • Analyze myelin formation and remyelination capacity

  • Examine Fas-Casp8-Bid-axis in mitochondria-associated apoptosis

• Demyelination models:

  • Lysolecithin-induced demyelination

  • Experimental autoimmune encephalomyelitis (EAE)

  • Compare wild-type vs. GSTA4-manipulated animals

  • Assess Caspase-8 activity in oligodendrocytes

• Therapeutic targeting approaches:

  • Test effects of dimethyl fumarate and clemastine fumarate on GSTA4 expression

  • Assess drug effects on oligodendrocyte survival and differentiation

  • Evaluate remyelination potential

GSTA4 has been identified as a mediator of oligodendrocyte survival during differentiation. GSTA4 overexpression reduces Fas expression and Casp8-Bid-axis activity in adult oligodendrocyte precursor cells, improving survival during differentiation, with effects confirmed in both lysolecithin-induced demyelination and EAE models .

How should I design experiments to study GSTA4 in skin cancer models?

GSTA4 has established roles in skin cancer susceptibility. For optimal experimental design:

• Two-stage skin carcinogenesis model:

  • Initiation: Single DMBA application

  • Promotion: Repeated TPA (12-O-tetradecanoylphorbol-13-acetate) treatment

  • Compare tumor development between wild-type and Gsta4-/- mice

  • Measure tumor multiplicity, size, and progression

• GSTA4 expression analysis:

  • Quantify mRNA using qPCR (primers: 5′-CAACCCGGAAGTCAGAGGAA-3′ and 5′-AGCACGCTGCACTAGAACTTCA-3′)

  • Normalize to appropriate housekeeping genes (e.g., Hras1)

  • Measure protein levels via Western blot and immunohistochemistry

  • Compare resistant (e.g., C57BL/6) vs. susceptible (e.g., DBA/2) mouse strains

• GSTA4 activity assays:

  • Spectrophotometric measurement of enzymatic activity

  • Assess activity in epidermal extracts

  • Compare baseline vs. post-TPA treatment

• Human relevance:

  • Analyze GSTA4 polymorphisms in human skin cancer samples

  • Conduct tag SNP-based case-control studies

  • Focus on basal cell and squamous cell carcinomas

Studies found Gsta4-deficient mice (C57BL/6.Gsta4-/-) were more sensitive to TPA skin tumor promotion (0.8 tumors per mouse vs. 0.4 tumors in wild-type controls; difference = 0.4 tumors per mouse; 95% CI = 0.1 to 0.7, p = 0.007). TPA induced GSTA4 mRNA and protein in resistant C57BL/6 mice but not susceptible DBA/2 mice .

What are the best approaches to investigate GSTA4's role in cisplatin-induced ototoxicity?

Cisplatin ototoxicity shows sex-dependent differences in GSTA4-mediated protection. For comprehensive investigation:

• Auditory function assessment:

  • Auditory brainstem response (ABR) testing

  • Compare threshold shifts between:

    • Wild-type vs. Gsta4-/- mice

    • Male vs. female subjects

    • Before and after cisplatin treatment

• Histological analysis:

  • Quantify spiral ganglion neuron (SGN) density in apical, middle, and basal cochlear regions

  • Measure stria vascularis thickness

  • Assess hair cell survival

  • Compare between genotypes and sexes

• 4-HNE accumulation:

  • Immunostain for 4-HNE-protein adducts

  • Quantify levels in cochlear structures

  • Correlate with functional outcomes

  • Compare male vs. female responses

• Hormonal influence studies:

  • Conduct ovariectomy experiments in female mice

  • Measure Gsta4 mRNA expression and protein levels

  • Assess Nrf2 expression (regulator of detoxification genes)

  • Evaluate estrogen's role in GSTA4-mediated protection

Research shows cisplatin-treated female Gsta4-/- mice display 34-41% decrease in SGN densities in apical and basal cochlear regions compared to wild-type females (p<0.01 and p<0.05, respectively). Female Gsta4-/- mice also showed 64% decrease in stria vascularis thickness in apical regions. These effects were not observed in male mice, suggesting sex-dependent GSTA4 protection .

How can I investigate GSTA4's tissue distribution in normal versus pathological states?

GSTA4 shows unique tissue distribution patterns that change during pathological conditions. For comprehensive analysis:

• Multi-tissue immunohistochemical mapping:

  • Develop tissue-specific staining protocols

  • Use specific anti-GSTA4 antibodies (e.g., against peptide corresponding to amino acids 209-222)

  • Compare normal tissues to pathological states (cirrhosis, UV-irradiated skin, myocardial infarction)

  • Evaluate cellular and subcellular localization

• Disease model comparison:

  • Cancer: Compare normal vs. tumor tissue (e.g., hepatocellular carcinoma)

  • Fibrosis: Analyze GSTA4 in normal vs. cirrhotic liver

  • Skin damage: Compare normal vs. UV-irradiated skin

  • Cardiac injury: Examine GSTA4 in normal vs. infarcted myocardium

• Cellular localization analysis:

  • Perform co-staining with cell type-specific markers

  • Use high-resolution confocal microscopy

  • Determine subcellular compartmentalization

  • Quantify expression levels by cell type

Research shows GSTA4 is localized in hepatocytes, bile duct cells, and vascular endothelial cells in liver; upper layers of keratinocytes and glands in skin; proximal tubules in kidney; epithelial and muscle cells in colon; muscle cells in heart; and neurons in brain. Expression increases in cirrhosis, UV-irradiated skin, and myocardial infarction, but decreases in hepatocellular carcinoma .

What experimental strategies reveal GSTA4's interactions with signaling pathways in cancer models?

GSTA4 modulates multiple cancer-related signaling pathways. For comprehensive analysis:

• Signaling node phosphorylation analysis:

  • Generate stable GSTA4-manipulated cell lines:

    • Knockdown: siRNA transfection (e.g., Santa Cruz Biotechnology sc-105424 with Lipofectamine-2000)

    • Overexpression: Retroviral vectors (e.g., pQCXIP-GSTA4, select with 0.5 μg/ml puromycin)

  • Assess phosphorylation status of key nodes:

    Pathway	Target	Phosphorylation Site
    AKT/mTOR	AKT	Ser473
    AKT/mTOR	mTOR	Ser2448
    GSK-3β	GSK-3β	Ser-9
    Adhesion	FAK	Tyr925
    Src	Src	Ser17
    MAPK	p38	Multiple sites

• Functional correlation:

  • Link pathway alterations to phenotypic changes:

    • Proliferation (EdU incorporation, MTT assays)

    • Invasion (Transwell assays)

    • Anchorage-independent growth (colony formation in semi-solid agar)

    • Apoptosis resistance (flow cytometry)

• Pathway inhibitor studies:

  • Use specific inhibitors for identified pathways

  • Determine if inhibitors rescue GSTA4-dependent phenotypes

  • Identify critical downstream mediators

• In vivo validation:

  • Xenograft models with GSTA4-manipulated cells

  • Analyze tumor growth kinetics

  • Assess pathway activation in tumor tissues

  • Test combinatorial approaches with pathway inhibitors

Studies demonstrate that inactivation of GSTA4 in HCT116 colorectal cancer cells blocks AKT and p38 MAPK pathways, leading to proliferative suppression both in vitro and in xenograft models. These findings establish GSTA4 as a regulator of key oncogenic signaling pathways .