CLCN5 Antibody

What is the cellular distribution of CLCN5 and what controls should I use for antibody validation?

CLCN5 is primarily expressed in renal proximal tubular cells, located mainly in subapical endosomes. It is also expressed in podocytes, α intercalated cells of the cortical collecting duct, and in the thick ascending limb of Henle's loop . When validating CLCN5 antibodies, consider these controls:

• Positive controls: MDCK cells and mouse brain tissue have shown reliable CLCN5 expression for Western blot

• Tissue controls: Human kidney tissue sections are ideal for immunohistochemistry validation

• Negative controls: Use CLCN5 knockdown cell lines created through shRNA (such as TRCN0000043903-907, TRCN000414058, and TCRN000427059)

• Cross-reactivity controls: Test for potential cross-reactivity with other CLC family members, particularly ClC-3 and ClC-4, which share approximately 66% epitope sequence similarity

What molecular weight should I detect when using CLCN5 antibodies in Western blot?

CLCN5 has two predominant splice proteoforms that researchers should be aware of:

Most commercially available antibodies detect a band between 80-90 kDa, corresponding to the shorter CLCN5 proteoform . When analyzing Western blot results, be aware that post-translational modifications may affect the apparent molecular weight.

What are the recommended protocols for CLCN5 antibody applications?

Based on experimental validations, the following protocols are recommended:

For optimal results, validate dilution ratios with your specific samples as expression levels may vary between tissues and experimental conditions.

How can I differentiate between wild-type and mutant CLCN5 proteins using immunofluorescence?

For comparative analysis of wild-type versus mutant CLCN5 localization:

• Generate expression constructs with wild-type CLCN5 (WT_CLCN5) and mutant CLCN5 (e.g., L521F_CLCN5) in a GFP-tagged vector system (such as pCMV6-AC-GFP)

• Transfect your cells of interest (HEK, HK-2, or human podocytes) using Lipofectamine 2000 (following manufacturer's protocol)

• Allow 48 hours for protein expression

• Fix cells with 4% paraformaldehyde in PBS

• Mount and image using confocal microscopy

Wild-type CLCN5 typically shows both cytoplasmic and cell surface distribution, while mutant forms (like L521F) predominantly display intracellular distribution . For colocalization studies with lysosomes, use Lysotracker deep red stain alongside GFP-tagged CLCN5 constructs to determine if mutant proteins are directed to lysosomes for degradation .

What methods should I use to study the role of CLCN5 in endocytosis?

To investigate CLCN5's role in endocytosis, implement the following transferrin uptake assay:

• Generate stable CLCN5 knockdown cell lines using lentiviral transduction of CLCN5-specific shRNA (validated shRNAs: TRCN0000043903-907)

• Confirm knockdown efficiency by Western blot

• Perform transferrin uptake assay:

  • Incubate cells with Alexa Fluor-conjugated Transferrin from Human Serum (e.g., Molecular Probes, Cat. No. T23365)

  • Remove surface-bound transferrin using citrate buffer (pH 2.5)

  • Collect images at 10, 15, and 30 minutes post-incubation using confocal microscopy

  • Quantify by counting cells with punctate transferrin labeling (positive) versus cells without distinct transferrin puncta (negative)

  • Analyze at least 50 cells per condition

This assay reveals functional endocytic defects associated with CLCN5 deficiency, as CLCN5 knockdown podocytes demonstrate significantly reduced transferrin uptake compared to control cells .

How can I evaluate the functional consequences of CLCN5 deficiency in podocytes?

To assess podocyte function following CLCN5 knockdown, employ these established assays:

Cell Proliferation Assay:

• Plate equal numbers (50,000 cells) of control and CLCN5 knockdown podocytes

• Allow growth for 24, 48, or 72 hours

• Trypsinize cells and count using a hemocytometer at each time point

• Plot cell numbers and calculate differences between control and knockdown clones

Migration/Wound Assay:

• Grow control and CLCN5 knockdown podocytes to confluence in 35-mm glass-bottom culture dishes

• Serum-starve cells in RPMI 1640 medium for 8-12 hours

• Create a scratch wound using a 1-10 μl pipette tip (two strokes at a 90-degree angle)

• Wash twice with PBS to remove suspended cells

• Culture in complete medium at 33°C for 12 hours

• Capture images at 0, 6, and 10 hours post-wounding

• Calculate migration rate using ImageJ software

CLCN5-deficient podocytes typically exhibit reduced proliferation and increased migration rates, which are considered indicators of podocyte injury .

What approaches can I use to distinguish between CLCN5 and other CLC family members in immunostaining?

Due to the high sequence homology between CLC family members, cross-reactivity is a significant concern when using CLCN5 antibodies. Implement this systematic approach:

• Perform immunolabeling for ClC-3, ClC-4, and ClC-5 on serial sections of the same sample

• Compare staining patterns, noting that:

  • Tubular apical staining is almost exclusively attributable to ClC-5

  • Cytoplasmic staining may represent ClC-3 and ClC-5, with minimal contribution from ClC-4

• Include appropriate controls:

  • Positive control: wild-type kidney tissue

  • Negative control: CLCN5 knockout tissue when available

  • Peptide competition assay to confirm antibody specificity

For definitive discrimination, consider using multiple antibodies targeting different epitopes of CLCN5, or implement genetic approaches such as CLCN5 knockdown with subsequent rescue experiments.

How can CLCN5 antibodies be used to investigate Dent disease mechanisms?

Dent disease type 1 results from mutations in the CLCN5 gene. To study disease mechanisms:

• Analyze ClC-5 expression patterns in kidney biopsies from Dent disease patients compared to controls:

  • In normal kidney, ClC-5 immunostaining appears mainly apical and subapical

  • In Dent disease type 1 biopsies, ClC-5 expression is typically reduced or absent

• Perform co-immunostaining for ClC-5 with megalin and cubilin (which are functionally related proteins in the endocytic pathway):

  • Megalin and cubilin expression is significantly reduced in Dent disease type 1 biopsies compared to controls

  • The reduction in ClC-5, megalin, and cubilin expression correlates with disease severity

• For functional analysis, compare wild-type and mutant CLCN5 proteins:

  • Transfect cells with wild-type and mutant CLCN5 constructs

  • Analyze cellular localization using immunofluorescence

  • Mutant CLCN5 proteins (e.g., L521F) often show aberrant cytoplasmic retention and increased lysosomal colocalization

What experimental approaches can detect CLCN5 in podocytes and evaluate its role in glomerular disease?

To investigate CLCN5's role in podocyte function and glomerular pathology:

• Confirm CLCN5 expression in podocytes:

  • Perform Western blot of cultured human podocyte lysates using anti-CLCN5 antibody

  • A single band at approximately 80 kDa typically represents the shorter CLCN5 proteoform

  • Verify with immunofluorescence microscopy of cultured podocytes

• Analyze glomerular CLCN5 expression in kidney sections:

  • Test normal mouse and human kidney sections

  • CLCN5 protein expression can be detected in glomeruli and parietal epithelial cells

  • Compare expression patterns between normal and diseased kidney samples

• Functional analysis through CLCN5 knockdown:

  • Generate stable CLCN5 knockdown podocyte cell lines

  • Assess endocytosis (transferrin uptake)

  • Evaluate cell proliferation and migration rates

  • Analyze collagen synthesis and regulation through the β-catenin pathway

CLCN5 knockdown in podocytes typically results in defective endocytosis, decreased proliferation, and increased migration, suggesting that CLCN5 mutations may contribute to glomerular pathology in Dent disease through direct effects on podocyte function .

How should I design experiments to study CLCN5 mutations and their effects on protein trafficking?

To investigate how CLCN5 mutations affect protein trafficking and function:

• Mutation analysis and prediction:

  • Use in silico prediction tools (PolyPhen-2, SIFT, MutationTaster) to predict the potential impact of specific mutations

  • For example, the L521F mutation has been predicted to be damaging with a PolyPhen-2 score of 0.884 (sensitivity: 0.82; specificity: 0.94)

• Expression construct preparation:

  • Generate expression vectors containing wild-type CLCN5 and mutant variants

  • Use a GFP-tagged vector system (e.g., pCMV6-AC-GFP) for visualization

• Cellular localization studies:

  • Transfect appropriate cell lines (HEK cells, HK-2 cells, or human podocytes)

  • Analyze subcellular distribution using confocal microscopy

  • Compare patterns between wild-type and mutant proteins

• Lysosomal colocalization:

  • Use Lysotracker deep red to stain lysosomes

  • Examine colocalization of GFP-labeled CLCN5 with lysosomal markers

  • Mutations like L521F typically show increased lysosomal colocalization compared to wild-type CLCN5

This approach allows determination of how specific mutations affect CLCN5 trafficking, which can provide insights into disease mechanisms and potential therapeutic strategies.

What are the optimal protocols for generating and validating CLCN5 knockdown models?

For reliable CLCN5 knockdown models:

• shRNA selection and validation:

  • Use validated CLCN5-specific shRNAs (e.g., TRCN0000043903-907, TRCN000414058, TCRN000427059)

  • Screen multiple shRNA constructs to identify those with highest knockdown efficiency

  • Transfect target cells using Lipofectamine 2000 according to manufacturer's protocol

• Selection of stable transfectants:

  • Grow transfected cells in medium containing 2.5 μg/ml puromycin

  • Maintain selection pressure to establish stable cell lines

• Knockdown verification:

  • Confirm CLCN5 knockdown by Western blot

  • Use anti-CLCN5 antibody (e.g., C1116, Sigma-Aldrich) at 1:500 dilution in TBST with 3% BSA

  • Include GAPDH (1:10,000) as loading control

  • Develop using Super Signal West Femto Maximum Sensitivity Substrate and analyze with an imaging system

• Functional validation:

  • Perform transferrin uptake assays to confirm functional consequences of CLCN5 knockdown

  • Assess cell proliferation and migration to evaluate cellular phenotype

What considerations are important when designing gene therapy experiments for CLCN5-related disorders?

When designing gene therapy approaches for conditions like Dent disease type 1:

• Vector selection and design:

  • Lentiviral vectors have been used successfully for CLCN5 gene delivery

  • Consider using tubule-specific promoters (e.g., Npt2a, Sglt2) to confine expression to tubular cells

• Treatment timing considerations:

  • Gene therapy performed in newborn ClC-5 null mice shows improved long-term transgene expression compared to treatment in adult mice

  • Early intervention may reduce immune responses against the therapeutic ClC-5 protein

• Outcome assessment:

  • Monitor transgene expression duration (effects may diminish over time due to immune responses)

  • Evaluate phenotypic improvements in disease markers (proteinuria, hypercalciuria)

  • Assess kidney function parameters

• Potential limitations:

  • Immune responses against the expressed ClC-5 protein may limit long-term efficacy

  • Cell-specific expression using tubule-specific promoters alone may not be sufficient to ameliorate all disease phenotypes

Early intervention appears to be a promising strategy to attenuate immune responses and achieve sustained therapeutic effects in CLCN5-related disorders.

How should I investigate the relationship between CLCN5 and collagen regulation in kidney cells?

Recent research has revealed that ClC-5 modulates collagen levels through the β-catenin pathway. To investigate this relationship:

• Collagen expression analysis:

  • Compare intracellular collagen levels between control, CLCN5 knockdown, and rescue (rWT) cells

  • Process cells for immunofluorescence using anti-collagen I/IV antibodies

  • CLCN5 knockdown cells typically show massive increases in intracellular collagen I and IV signals

• Extracellular collagen assessment:

  • Analyze extracellular collagen by fixing non-permeabilized cells

  • Use confocal microscopy to visualize and quantify extracellular collagen deposition

• Pathway analysis:

  • Investigate the β-catenin pathway as a mediator of CLCN5's effects on collagen synthesis

  • Examine whether CLCN5 knockdown affects β-catenin signaling components

  • Test whether β-catenin pathway modulators can rescue the collagen phenotype in CLCN5-deficient cells