Phospho-HNRNPC (Ser260) Antibody

What is the specificity of Phospho-HNRNPC (Ser260) antibody?

Phospho-HNRNPC (Ser260) antibody specifically recognizes the Heterogeneous Nuclear Ribonucleoproteins C1/C2 (HNRNPC) protein when phosphorylated at serine 260. The antibody binds to the amino acid region 231-280 of human HNRNP C1/C2 and detects endogenous levels of this protein only when the Ser260 residue is phosphorylated . Specificity can be verified through blocking experiments with the phospho-peptide, as shown in Western blot analyses where antibody binding is abolished when pre-absorbed with the immunogen peptide . This high specificity makes the antibody valuable for discriminating between phosphorylated and non-phosphorylated forms of HNRNPC in experimental settings.

What are the recommended applications for Phospho-HNRNPC (Ser260) antibody?

Phospho-HNRNPC (Ser260) antibody has been validated for multiple research applications with specific recommended dilution ranges:

These applications enable researchers to detect phosphorylated HNRNPC in various experimental contexts, from protein expression analysis to cellular localization studies . The antibody has been particularly well-characterized for Western blot analysis of cell lysates treated with H₂O₂, where it successfully detects the phosphorylated form of HNRNPC at approximately 41 kDa (observed molecular weight), differing slightly from the calculated molecular weight of 34 kDa .

What is the biological significance of HNRNPC phosphorylation at Ser260?

HNRNPC phosphorylation at Ser260 appears to play a regulatory role in its function as an RNA-binding protein. HNRNPC is involved in several critical cellular processes, including:

• Nucleation of 40S hnRNP particle assembly after binding pre-mRNA

• Modulation of mRNA stability through interaction with poly-U tracts in 3'-UTR or 5'-UTR regions

• Regulation of translation levels of bound mRNA molecules

• Participation in early spliceosome assembly and pre-mRNA splicing

• Involvement in the "m6A-switch" mechanism that regulates mRNA splicing

Phosphorylation at Ser260 likely influences these functions by altering HNRNPC's binding affinity to RNA or its interactions with other proteins. This post-translational modification has been implicated in cellular responses to stress conditions, as demonstrated in studies where H₂O₂ treatment (100μM for 15 minutes) increased HNRNPC phosphorylation at Ser260 . Understanding this phosphorylation event provides insights into how RNA metabolism and gene expression are regulated under various physiological and pathological conditions.

How can I optimize nuclear extraction protocols for detecting phosphorylated HNRNPC?

Optimizing nuclear extraction is critical for studying phosphorylated HNRNPC since this protein primarily localizes to the nucleus. A methodological approach includes:

• Cell preparation: Culture cells to 80-90% confluence before extraction.

• Extraction protocol selection: Use commercial kits like the Minute™ Cytoplasmic and Nuclear Fractionation kit, which has been validated for phospho-HNRNPC detection . Alternatively, employ a modified Dignam protocol:

  • Wash cells twice with ice-cold PBS

  • Resuspend pellet in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl)

  • Incubate on ice for 15 minutes

  • Add NP-40 to 0.6% final concentration

  • Vortex for 10 seconds and centrifuge at 10,000 × g for 30 seconds

  • Extract nuclear proteins with high-salt buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol)

• Phosphatase inhibition: Critical for preserving phosphorylation status - include sodium orthovanadate (1 mM), sodium fluoride (10 mM), β-glycerophosphate (5 mM), and phosphatase inhibitor cocktails in all buffers.

• Protein quantification: Use Bradford or BCA assays to normalize loading for Western blot analysis.

Western blot analysis has successfully detected phospho-HNRNPC in nuclear extracts from 293 cells, confirming this approach's efficacy . This optimization ensures reliable detection of phosphorylated HNRNPC while minimizing cytoplasmic contamination and phosphatase activity.

What are the known stimuli that induce HNRNPC Ser260 phosphorylation?

Several stimuli have been documented to induce HNRNPC Ser260 phosphorylation:

• Oxidative stress: H₂O₂ treatment (100μM for 15 minutes) consistently increases Ser260 phosphorylation in 293 cells, as demonstrated in Western blot analyses . This suggests HNRNPC phosphorylation may be part of the cellular response to reactive oxygen species.

• DNA damage: While not explicitly shown in the search results, the phosphorylation of RNA-binding proteins like HNRNPC often occurs during DNA damage responses, potentially linking it to cellular stress adaptation mechanisms.

• Cell cycle regulation: HNRNPC phosphorylation states may vary throughout the cell cycle, affecting its RNA-binding properties and subcellular localization.

To experimentally induce and detect HNRNPC phosphorylation, researchers should:

• Treat cells with appropriate stimuli (e.g., H₂O₂) using optimized dose and time parameters

• Include appropriate controls (untreated cells, phosphatase-treated extracts)

• Use phospho-specific antibodies in combination with total HNRNPC antibodies to calculate relative phosphorylation levels

• Consider phospho-enrichment strategies for low-abundance detection

This approach enables researchers to study the dynamics of HNRNPC phosphorylation in response to various cellular conditions.

How does phosphorylation at Ser260 affect HNRNPC's RNA-binding properties?

Phosphorylation at Ser260 likely modulates HNRNPC's interaction with target RNAs, though the precise mechanisms require further research. Based on current understanding:

• Binding affinity modulation: Phosphorylation may alter the protein's affinity for poly-U tracts in the 3'-UTR or 5'-UTR of mRNA molecules. HNRNPC tetramers typically bind 230-240 nucleotides, while trimers of HNRNPC tetramers bind approximately 700 nucleotides . Phosphorylation could affect these binding capacities.

• m6A-switch mechanism: HNRNPC is known to participate in a mechanism where N6-methyladenosine (m6A) modifications alter local RNA structure, facilitating HNRNPC binding and regulating mRNA splicing . Phosphorylation at Ser260 potentially influences this interaction.

• Protein-protein interactions: Phosphorylation could affect HNRNPC's interactions with other components of the splicing machinery or hnRNP particles.

To experimentally investigate these effects, researchers should consider:

• RNA immunoprecipitation (RIP) assays comparing wild-type and phospho-mimetic (S260D) or phospho-deficient (S260A) HNRNPC mutants

• in vitro RNA binding assays with purified phosphorylated and non-phosphorylated proteins

• CLIP-seq (Crosslinking and immunoprecipitation followed by sequencing) to identify differential RNA targets

These approaches would help elucidate how Ser260 phosphorylation functionally impacts HNRNPC's role in RNA metabolism and processing.

What is the optimal protocol for using Phospho-HNRNPC (Ser260) antibody in Western blot analysis?

The following protocol has been validated for Western blot detection of phosphorylated HNRNPC:

• Sample preparation:

  • Extract nuclear proteins from cells (see question 2.1)

  • Include phosphatase inhibitors in all buffers

  • Load 20-40 μg of nuclear protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane (0.45 μm)

• Blocking and antibody incubation:

  • Block membrane with 5% BSA in TBST for 1 hour at room temperature

  • Incubate with Phospho-HNRNPC (Ser260) antibody at 1:500-1:2000 dilution in blocking buffer overnight at 4°C

  • Wash 3× with TBST, 10 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 3× with TBST, 10 minutes each

• Detection and controls:

  • Develop using ECL substrate

  • Include positive controls (H₂O₂-treated 293 cells) showing a band at approximately 41 kDa

  • Include specificity controls (antibody pre-absorbed with phospho-peptide)

• Validation strategies:

  • Compare with non-phosphorylated HNRNPC detection

  • Use lambda phosphatase treatment as a negative control

  • Consider subcellular fractionation to enrich nuclear proteins

This methodology has successfully detected phospho-HNRNPC in cell lysates, showing increased phosphorylation following H₂O₂ treatment , confirming the protocol's reliability for studying this post-translational modification.

How can I optimize immunohistochemistry protocols for detecting phospho-HNRNPC in tissue sections?

For optimal immunohistochemical detection of phospho-HNRNPC in tissue sections:

• Tissue preparation:

  • Use formalin-fixed paraffin-embedded (FFPE) tissue sections (4-6 μm thick)

  • Deparaffinize sections through xylene and graded alcohol series

• Antigen retrieval - critical step:

  • Use high-pressure and high-temperature Tris-EDTA buffer (pH 8.0)

  • Alternative: citrate buffer (pH 6.0) with pressure cooker treatment

  • Cool sections to room temperature before proceeding

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5-10% normal serum in PBS for 1 hour

  • Incubate with Phospho-HNRNPC (Ser260) antibody at 1:100 dilution overnight at 4°C

  • Wash 3× with PBS, 5 minutes each

  • Apply appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

• Detection and controls:

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Include negative controls: primary antibody omission and antibody pre-absorbed with immunogen peptide

  • Include positive control tissues (human brain has been validated)

• Evaluation parameters:

  • Score staining intensity (0-3+)

  • Assess cellular and subcellular localization (primarily nuclear expected)

  • Quantify percentage of positive cells

This protocol has been validated for human brain tissue, confirming its efficacy for detecting phospho-HNRNPC in tissue sections . Proper antigen retrieval is particularly critical for exposing phosphorylated epitopes in FFPE tissues.

What are the recommended approaches for using Cell-Based ELISA to quantify HNRNPC phosphorylation?

Cell-Based ELISA provides a high-throughput method for quantifying HNRNPC phosphorylation directly in cultured cells without the need for lysate preparation. For optimal results:

• Cell preparation:

  • Seed cells in 96-well plates (recommended >5000 cells/well)

  • Allow attachment and growth to desired confluence

  • Apply experimental treatments (e.g., H₂O₂, kinase inhibitors)

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 20 minutes at room temperature

  • Wash gently with PBS

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

• Antibody incubation:

  • Block with 5% BSA in PBS for 1 hour

  • Incubate with Phospho-HNRNPC (Ser260) antibody at appropriate dilution overnight at 4°C

  • Wash 3× with PBS

  • Incubate with HRP-conjugated secondary antibody

• Detection and normalization:

  • Develop with TMB substrate and read absorbance at 450 nm

  • Normalize to cell number using crystal violet staining or a total protein antibody

  • Calculate relative phosphorylation by comparison to total HNRNPC levels

• Experimental design considerations:

  • Include time-course and dose-response experiments

  • Use phosphatase treatments as negative controls

  • Consider dual detection of phosphorylated and total HNRNPC

The hnRNP C1/2 (Phospho-Ser260) Colorimetric Cell-Based ELISA Kit offers a standardized approach, with sensitivity to detect phosphorylation in samples with >5000 cells . This method allows for efficient screening of compounds that may affect HNRNPC phosphorylation status.

How should I interpret discrepancies between observed and calculated molecular weights for phospho-HNRNPC?

Researchers frequently observe a discrepancy between the calculated molecular weight of HNRNPC (34 kDa) and its apparent molecular weight on Western blots (41 kDa) . This difference requires careful interpretation:

• Causes of migration differences:

  • Post-translational modifications: Phosphorylation itself adds minimal mass but can significantly alter protein migration by changing charge distribution

  • Alternative splicing: HNRNPC exists in two isoforms (C1 and C2) resulting from alternative splicing

  • Other modifications: Potential glycosylation, sumoylation, or ubiquitination

• Verification approaches:

  • Isoform-specific detection: Use antibodies that distinguish between C1 and C2 isoforms

  • Phosphatase treatment: Lambda phosphatase treatment should reduce the molecular weight if phosphorylation causes the shift

  • Mass spectrometry: Definitive identification of protein and modifications

• Interpretation guidelines:

  • A consistent observation of 41 kDa across studies indicates this is the true migration pattern for phosphorylated HNRNPC

  • Compare with literature reports of HNRNPC migration patterns

  • Document all experimental conditions that might affect migration (buffer systems, gel percentage)

This molecular weight discrepancy is a documented characteristic of phospho-HNRNPC detection and should not be interpreted as non-specific binding . Researchers should report both calculated and observed molecular weights in their publications to avoid confusion.

What strategies can address weak or inconsistent phospho-HNRNPC signal in Western blots?

When encountering weak or inconsistent phospho-HNRNPC signals:

• Sample preparation optimization:

  • Phosphatase inhibition: Ensure fresh phosphatase inhibitors in all buffers

  • Rapid processing: Minimize time between cell harvesting and protein denaturation

  • Nuclear enrichment: Use subcellular fractionation to concentrate HNRNPC

  • Protein loading: Increase to 40-50 μg per lane for low abundance phospho-proteins

• Technical adjustments:

  • Antibody concentration: Increase primary antibody concentration to 1:500

  • Incubation time: Extend to overnight at 4°C

  • Detection system: Use high-sensitivity chemiluminescent substrates or signal amplification systems

  • Membrane: PVDF may retain small proteins better than nitrocellulose

• Experimental design improvements:

  • Positive controls: Include H₂O₂-treated 293 cells as validated positive controls

  • Phospho-enrichment: Consider phospho-protein enrichment columns

  • Stimulation optimization: Titrate stimulation conditions (concentration and time)

• Validation approaches:

  • Alternative antibodies: Test multiple phospho-specific antibodies if available

  • Phospho-mimetic mutants: Use S260D HNRNPC as positive controls

  • Phosphatase treatment: Include as negative control

These strategies address common issues with phospho-protein detection, with particular attention to preserving the phosphorylation status during sample preparation, a critical factor for successful phospho-HNRNPC detection.

How can I differentiate between specific and non-specific binding in immunohistochemistry with phospho-HNRNPC antibody?

Distinguishing specific from non-specific binding in phospho-HNRNPC immunohistochemistry requires several validation strategies:

• Essential controls:

  • Peptide competition: Pre-absorb antibody with immunogen peptide; this should eliminate specific staining as demonstrated in human brain tissue sections

  • Phosphatase treatment: Treat serial sections with lambda phosphatase to remove phosphate groups

  • Primary antibody omission: Replace with same-species IgG at equivalent concentration

• Staining pattern evaluation:

  • Subcellular localization: Specific phospho-HNRNPC staining should be predominantly nuclear

  • Cell type specificity: Consider known expression patterns of HNRNPC across tissue types

  • Signal-to-noise ratio: Compare staining intensity between positive cells and background

• Optimization strategies:

  • Antibody titration: Test serial dilutions (1:50, 1:100, 1:300) to determine optimal concentration

  • Antigen retrieval methods: Compare different buffers and conditions

  • Blocking optimization: Increase blocking agent concentration or time to reduce background

• Advanced validation:

  • Double immunofluorescence: Co-stain with total HNRNPC antibody to confirm colocalization

  • Correlation with phosphorylation-inducing treatments: Compare tissues from control and treated conditions

  • Genetic models: Use tissues from knockout models as negative controls where available

These approaches, particularly the peptide competition assay which has been validated for this antibody , provide robust methods to confirm specificity of phospho-HNRNPC staining in tissue sections.

What considerations are important when designing experiments to study the functional consequences of HNRNPC Ser260 phosphorylation?

When designing experiments to investigate functional consequences of HNRNPC Ser260 phosphorylation:

• Genetic manipulation approaches:

  • Phospho-mimetic mutants: Generate S260D (mimics phosphorylation) and S260A (prevents phosphorylation) mutants

  • Expression systems: Use inducible expression to control timing and levels

  • CRISPR-Cas9 knock-in: Create cell lines with endogenous phospho-mutants

• Functional readouts to assess:

  • RNA binding: RNA immunoprecipitation followed by qPCR or sequencing

  • Protein interactions: Co-immunoprecipitation with known HNRNPC partners

  • Splicing regulation: Exon-specific RT-PCR for known targets

  • mRNA stability: Actinomycin D chase experiments with target mRNAs

  • Subcellular localization: Immunofluorescence under different conditions

• Physiological context considerations:

  • Stress conditions: Test oxidative stress (H₂O₂), which is known to induce phosphorylation

  • Cell cycle phases: Synchronize cells and analyze phase-specific effects

  • Tissue-specific differences: Compare effects in different cell types

• Integration with other approaches:

  • Phosphoproteomics: Identify other modifications that may cross-talk with Ser260

  • Transcriptomics: RNA-seq to identify global effects on gene expression

  • Structure-function analysis: Consider how phosphorylation affects protein structure

This comprehensive experimental design approach enables researchers to establish causal relationships between HNRNPC Ser260 phosphorylation and its biological functions, particularly in RNA metabolism and gene expression regulation, which are central to HNRNPC's cellular roles .