SMARCAL1 Antibody, FITC conjugated

What is SMARCAL1 and what cellular functions does it regulate?

SMARCAL1 is a DNA translocase and ATP-dependent annealing helicase that functions in multiple cellular processes. Unlike typical helicases that unwind DNA, SMARCAL1 catalyzes the reannealing of single-stranded regions in DNA that are bound by Replication Protein A (RPA) .

SMARCAL1 serves several critical functions:

• DNA damage response: SMARCAL1 is phosphorylated by DNA repair kinases (ATM, ATR, DNA-PK) during replication stress and helps stabilize stalled replication forks .

• Transcriptional regulation: SMARCAL1 occupies chromatin regions enriched for histone H3 lysine 4 tri-methylation (H3K4me3), suggesting a role in transcriptional regulation .

• Immune regulation: Recent research has identified SMARCAL1 as a dual regulator of innate immune signaling and PD-L1 expression, with potential implications for cancer immunotherapy .

Mutations in SMARCAL1 cause Schimke immuno-osseous dysplasia (SIOD), a multisystem disorder characterized by spondyloepiphyseal dysplasia, renal dysfunction, immunodeficiency, and neurological impairment .

How do FITC-conjugated SMARCAL1 antibodies differ from unconjugated variants in experimental applications?

FITC-conjugated SMARCAL1 antibodies offer several methodological advantages over unconjugated variants:

• Direct visualization: The FITC fluorophore allows direct detection without secondary antibodies, simplifying workflows and reducing background in immunofluorescence experiments .

• Multicolor analysis: FITC excitation/emission spectra (495/519 nm) complement other fluorophores, enabling multiplexed analysis with differently labeled antibodies against interacting proteins or cellular structures .

• Flow cytometry capability: FITC-conjugated antibodies are particularly useful for flow cytometry applications to quantify SMARCAL1 expression levels across cell populations .

• Application restrictions: Some FITC-conjugated antibodies may have more limited validated applications compared to unconjugated versions. For example, while some unconjugated SMARCAL1 antibodies are validated for WB, IF, IP, and ELISA, FITC variants may be primarily optimized for IF and flow cytometry .

• Photostability challenges: FITC is more susceptible to photobleaching than some alternative fluorophores, requiring careful imaging protocols.

What are optimal immunofluorescence protocols for SMARCAL1 FITC-conjugated antibodies?

When performing immunofluorescence with FITC-conjugated SMARCAL1 antibodies, follow these methodological guidelines:

Sample preparation:

• Fix cells with 4% paraformaldehyde (10-15 minutes) or 100% methanol (-20°C, 10 minutes) depending on epitope accessibility

• Permeabilize with 0.1-0.5% Triton X-100 in PBS (5-10 minutes)

• Block with 1-5% BSA or normal serum in PBS (30-60 minutes)

Antibody incubation:

• Dilute FITC-conjugated SMARCAL1 antibody according to manufacturer recommendations (typically 1:50-1:200)

• Incubate at 4°C overnight or at room temperature for 1-2 hours

• Wash extensively (3-5 times, 5 minutes each) with PBS containing 0.1% Tween-20

Counterstaining and mounting:

• Counterstain nuclei with DAPI (1:1000 dilution, 5 minutes)

• Mount with anti-fade mounting medium to minimize photobleaching

Critical considerations:

• SMARCAL1 typically shows nuclear localization with discrete foci formation after DNA damage

• When studying replication stress, synchronize cells and treat with appropriate agents (e.g., hydroxyurea) before fixation

• Include appropriate controls (primary antibody omission, isotype control, SMARCAL1-depleted cells)

How can SMARCAL1 FITC-conjugated antibodies be validated for specificity and functionality?

Rigorous validation is essential for reliable SMARCAL1 antibody experiments. Implement these methodological approaches:

Specificity validation:

• Western blot correlation: Confirm the antibody detects a single band at approximately 105-150 kDa (depending on species and post-translational modifications)

• siRNA/CRISPR depletion: Verify signal reduction in SMARCAL1-depleted cells

• Immunoprecipitation: If applicable, confirm the antibody can immunoprecipitate SMARCAL1 protein that is subsequently detected by other validated antibodies

Functionality testing:

• Nuclear localization: Verify predominantly nuclear staining pattern in immunofluorescence

• DNA damage response: Confirm increased SMARCAL1 foci formation after replication stress (e.g., hydroxyurea treatment)

• Chromatin association: Validate increased chromatin fractionation of SMARCAL1 after replication stress

Cross-reactivity assessment:

• Test the antibody against potential cross-reactive proteins, particularly other SMARCA family members

• Evaluate using multiple cell types to confirm consistent detection patterns

What techniques can be used to study SMARCAL1 localization at replication forks?

Several specialized techniques can examine SMARCAL1 localization at stalled replication forks:

SIRF (in situ proximity ligation assay for replisomes) assay:

• Pulse label cells with EdU to mark replication forks

• Fix and perform click chemistry to conjugate biotin to EdU

• Perform proximity ligation between FITC-conjugated SMARCAL1 antibody and anti-biotin antibody

• Quantify PLA spots to measure SMARCAL1 association with active replication sites

This approach has demonstrated that SMARCAL1 recruitment to replication forks increases after hydroxyurea treatment, as expected for a fork reversal factor .

Chromatin fractionation and immunoblotting:

• Fractionate cells into cytoplasmic, nucleoplasmic, and chromatin-bound fractions

• Analyze by Western blot for SMARCAL1 enrichment in chromatin fraction

• Compare untreated versus replication stress conditions

Research has shown that treatment with hydroxyurea for 4 hours increases the fraction of chromatin-associated SMARCAL1 in wild-type cells, confirming its role in the replication stress response .

Immunofluorescence co-localization with fork markers:

• Co-stain cells with FITC-conjugated SMARCAL1 antibody and antibodies against fork components (e.g., PCNA, RPA)

• Analyze co-localization using confocal microscopy and quantitative image analysis

How does SMARCAL1 expression correlate with immune infiltration in cancer?

Recent research has revealed important correlations between SMARCAL1 expression and immune profiles in cancer:

SMARCAL1 expression and leukocyte infiltration:

• Pan-cancer analysis shows that tumors with low SMARCAL1 expression exhibit upregulation of inflammatory response pathways

• In 53% of tumor types, the ratio of leukocyte score between SMARCAL1-low and SMARCAL1-high groups was greater than 1

• A positive correlation (R=0.68) exists between inflammatory response signatures and leukocyte infiltration in SMARCAL1-low tumors

Immunotherapy response correlation:

• Analysis of datasets from cancer patients treated with anti-PD-1 therapy revealed that responders expressed significantly lower levels of SMARCAL1 compared to non-responders

• SMARCAL1-deficient cells show reduced PD-L1 levels, potentially making them more susceptible to immune attack

These findings suggest SMARCAL1 may influence tumor immune evasion and could be a potential therapeutic target to enhance immunotherapy efficacy.

What experimental designs can investigate SMARCAL1's role in PD-L1 regulation?

To study SMARCAL1's influence on PD-L1 expression, consider these methodological approaches:

CRISPR-Cas9 knockout and PD-L1 assessment:

• Generate SMARCAL1-knockout cell lines using CRISPR-Cas9

• Measure PD-L1 protein levels via flow cytometry and Western blotting

• Assess PD-L1 mRNA levels using RT-qPCR

• Test response to known PD-L1 inducers (IFN-β, IFN-γ, EGF) in SMARCAL1-proficient versus deficient cells

Research using this approach has demonstrated that SMARCAL1-deficient cells exhibit reduced PD-L1 levels by both immunoblotting and flow cytometry .

ChIP-seq and CUT&RUN analysis:

• Perform chromatin immunoprecipitation with SMARCAL1 antibodies

• Analyze genome-wide chromatin occupancy patterns

• Look for enrichment at PD-L1 locus or regulatory elements

SMARCAL1 CUT&RUN studies have shown that it largely occupies chromatin regions enriched for H3K4me3, a mark of active promoters .

Transcription factor identification:

• Conduct FACS-based CRISPR-Cas9 screens in SMARCAL1-knockout and control cells

• Sort cells based on PD-L1 staining

• Determine sgRNA composition in different populations by sequencing

• Identify transcription factors that influence SMARCAL1-dependent PD-L1 regulation

This approach identified fifteen transcription factors whose sgRNAs were enriched in PD-L1-low populations of SMARCAL1-proficient but not -deficient cells .

How can I optimize detection of SMARCAL1 during DNA replication stress studies?

To maximize detection sensitivity of SMARCAL1 during replication stress experiments:

Cell synchronization and treatment optimization:

• Synchronize cells using double thymidine block or serum starvation

• Induce replication stress with hydroxyurea (typical concentration: 2-4 mM for 2-4 hours)

• Monitor SMARCAL1 recruitment kinetics by collecting samples at multiple timepoints

Detergent-resistant focus detection:

• Use a pre-extraction step (0.5% Triton X-100 in PBS for 5-10 minutes on ice) before fixation

• This removes soluble nuclear proteins and enhances visualization of chromatin-bound SMARCAL1

• Fix with 4% paraformaldehyde and proceed with standard immunofluorescence protocol

Signal amplification strategies:

• Consider tyramide signal amplification for enhanced sensitivity

• Use enhanced detection systems for chromatin-bound proteins

• Optimize imaging parameters (exposure time, gain, laser power) for FITC-conjugated antibodies

Quantitative analysis approaches:

• Count SMARCAL1 foci per nucleus

• Measure foci intensity

• Determine percentage of cells with >5-10 foci

• Compare before and after replication stress induction

Research has shown that accumulation of detergent-resistant SMARCAL1 nuclear foci is greatly reduced in hydroxyurea-treated cells with dysfunctional MUS81, indicating the importance of proper experimental controls .

What approaches can be used to study SMARCAL1 in fork reversal?

To investigate SMARCAL1's role in replication fork reversal, consider these methodological approaches:

RuvA chromatin association assay:

• Express GFP-fused RuvA (a four-way junction-binding factor) in cells

• Induce replication fork stalling (e.g., hydroxyurea treatment)

• Assess RuvA chromatin association as a proxy for reversed fork abundance

• Compare cells with normal versus altered SMARCAL1 function

Research using this approach demonstrated that cells with deregulated fork processing show reduced normalized amounts of chromatin-associated RuvA after hydroxyurea treatment, indicating fewer reversed forks .

Proximity ligation assays (PLA):

• Perform PLA between SMARCAL1 and RAD51 or other fork reversal factors

• Quantify PLA spots as a measure of protein-protein interactions at forks

• Compare under normal conditions versus replication stress

Electron microscopy:

• Perform DNA spreading and platinum shadowing

• Visualize directly reversed forks by electron microscopy

• Quantify frequency of reversed forks in SMARCAL1-proficient versus deficient cells

DNA fiber analysis:

• Label replication tracks with nucleoside analogs (CldU followed by IdU)

• Induce replication stress during the second label

• Spread DNA fibers and immunolabel the analogs

• Measure IdU/CldU ratio to assess fork protection

• Compare SMARCAL1-proficient versus deficient cells

How can SMARCAL1's chromatin occupancy be studied in relation to its dual functions?

To investigate SMARCAL1's chromatin association patterns related to both DNA repair and transcriptional functions:

CUT&RUN protocol optimization:

• Cross-link cells with formaldehyde (1%, 10 minutes at room temperature)

• Permeabilize and immobilize cells on ConA-coated beads

• Incubate with SMARCAL1 antibody (primary incubation time: 2 hours at room temperature)

• Add protein A-MNase fusion protein

• Activate MNase with calcium to cleave DNA around binding sites

• Extract and sequence DNA fragments

Histone mark co-localization analysis:

• Perform sequential or parallel CUT&RUN with antibodies against:

  • SMARCAL1

  • H3K4me3 (active promoters)

  • γH2AX (DNA damage sites)

• Compare binding profiles to distinguish transcriptional versus DNA repair functions

Research has shown that SMARCAL1 largely occupies chromatin regions enriched for H3K4me3, suggesting a significant role in transcriptional regulation .

Integration with functional genomics:

• Combine chromatin occupancy data with transcriptome analysis (RNA-seq)

• Compare gene expression changes in SMARCAL1-deficient cells with SMARCAL1 binding sites

• Categorize genes based on their dependency on SMARCAL1 for proper expression

How can I design experiments to investigate SMARCAL1's impact on immunotherapy response?

To study SMARCAL1's influence on immunotherapy efficacy, consider these experimental approaches:

Cancer organoid and co-culture systems:

• Generate SMARCAL1-knockout versus wild-type cancer organoids

• Co-culture with immune cells (T cells, NK cells)

• Treat with immune checkpoint inhibitors

• Assess tumor cell killing, immune cell activation, and cytokine profiles

Syngeneic mouse tumor models:

• Establish SMARCAL1-knockout versus control cancer cell lines

• Implant into immunocompetent mice

• Treat with immune checkpoint inhibitors

• Monitor tumor growth, immune infiltration, and survival

Human cancer sample analysis:

• Collect tumor samples from patients treated with immunotherapy

• Assess SMARCAL1 expression levels by immunohistochemistry or RNA-seq

• Correlate with treatment response, immune infiltration, and PD-L1 expression

Analysis of datasets from cancer patients treated with anti-PD-1 therapy has already revealed that responders express significantly lower levels of SMARCAL1 compared to non-responders, suggesting SMARCAL1 as a potential predictive biomarker for immunotherapy response .

Mechanistic studies:

• Assess changes in antigen presentation, MHC expression, and immunogenic signaling in SMARCAL1-deficient cells

• Investigate inflammatory cytokine production and response

• Examine changes in immune checkpoint receptor/ligand expression beyond PD-L1

What methodological considerations are important when studying SMARCAL1 in non-cycling cells?

Investigating SMARCAL1 in non-cycling cells presents unique challenges and requires specific methodological approaches:

Cell cycle arrest protocols:

• Serum starvation (0.1% FBS for 24-48 hours) to induce G0/G1 arrest

• Contact inhibition for confluent cells

• Use of CDK inhibitors like palbociclib for G1 arrest

• Verification of arrest by flow cytometry with propidium iodide or EdU staining

Fucci reporter system:

• Utilize Fucci (Fluorescence Ubiquitination Cell Cycle Indicator) cells to distinguish cell cycle phases

• Isolate G1 cells based on fluorescent markers

• Compare SMARCAL1 functions between cycling and non-cycling populations

Non-cycling cell analysis approaches:

• Monitor SMARCAL1 protein and mRNA levels in serum-starved versus proliferating cells

• Assess SMARCAL1 chromatin occupancy in G0/G1-arrested cells

• Compare DNA damage response functions in cycling versus non-cycling cells

Research has demonstrated that SMARCAL1 depletion reduces PD-L1 protein and mRNA levels in serum-starved cells, indicating that SMARCAL1 regulates PD-L1 in both cycling and non-cycling cells .

What techniques can differentiate between SMARCAL1's roles in DNA repair versus transcriptional regulation?

To distinguish between SMARCAL1's dual functions:

Domain-specific mutants:

• Generate cells expressing SMARCAL1 with mutations in specific functional domains:

  • ATPase domain mutants (affect DNA remodeling)

  • RPA-binding domain mutants (impair recruitment to stalled forks)

  • DNA-binding domain mutants (alter chromatin interaction)

• Compare effects on DNA repair versus gene expression

Conditional degradation approaches:

• Utilize auxin-inducible degron (AID) or dTAG systems for rapid SMARCAL1 depletion

• Monitor immediate (likely DNA repair-related) versus delayed (potentially transcriptional) phenotypes

Separation of function experiments:

• Identify conditions that predominantly activate one function:

  • Replication stress agents (hydroxyurea, aphidicolin) for DNA repair function

  • Specific transcriptional activators/repressors for gene regulatory function

• Compare SMARCAL1 localization, interaction partners, and downstream effects

ChIP-seq versus DNA damage co-localization:

• Compare genome-wide binding maps with localization to sites of DNA damage

• Analyze overlap and distinct binding patterns

• Correlate with gene expression changes and DNA repair outcomes

How can I analyze SMARCAL1's association with transcription factors in cancer?

To investigate SMARCAL1's interactions with transcription factors in cancer contexts:

Co-immunoprecipitation strategies:

• Immunoprecipitate SMARCAL1 using validated antibodies

• Probe for associated transcription factors identified in screening approaches

• Confirm interactions by reciprocal immunoprecipitation

• Compare interaction profiles between normal and cancer cells

ChromVAR and TOBIAS analysis:

• Perform ATAC-seq in SMARCAL1-proficient and deficient cells

• Apply ChromVAR analysis to identify transcription factor motifs affected by SMARCAL1 loss

• Use TOBIAS framework to predict transcription factors with altered chromatin binding

• Validate predictions with ChIP-seq or CUT&RUN

Research using these approaches identified 142 transcription factors whose binding to chromatin was potentially affected by SMARCAL1 loss .

Functional validation:

• Deplete identified transcription factors in SMARCAL1-proficient versus deficient cells

• Assess effects on target gene expression (e.g., PD-L1)

• Perform rescue experiments to establish hierarchy of regulation

A FACS-based CRISPR-Cas9 screen targeting 142 transcription factors identified fifteen TFs whose sgRNAs were enriched in the PD-L1-low population of SMARCAL1-proficient but not -deficient cells, suggesting their involvement in SMARCAL1-dependent PD-L1 regulation .

What is the relationship between SMARCAL1 and cancer prognosis across different tumor types?

Comprehensive pan-cancer analyses have revealed complex relationships between SMARCAL1 expression and clinical outcomes:

Expression patterns across cancer types:

• SMARCAL1 is overexpressed in most tumor types compared to normal tissues

• Particularly high expression observed in Glioma, Lung Adenocarcinoma (LUAD), Kidney Renal Clear Cell Carcinoma (KIRC), and Liver Hepatocellular Carcinoma (LIHC)

Prognostic correlations:

• Elevated SMARCAL1 linked to poor outcomes in Glioma, LUAD, and LIHC

• Counterintuitively, higher SMARCAL1 correlates with better survival in KIRC

• Progression-free survival is shorter for SMARCAL1-high patients in certain cancer types

Molecular pathways affected:

• SMARCAL1-low tumors show downregulation of cell proliferation pathways, c-Myc activation, TGF-β signaling, and DNA repair

• SMARCAL1-low tumors exhibit upregulation of inflammatory response pathways

• PD-L1 expression is significantly downregulated in 85% of tumor types in the SMARCAL1-low group

Immunotherapy response correlation:

• Analysis of datasets from immunotherapy-treated patients shows responders express lower SMARCAL1 levels compared to non-responders

• This suggests SMARCAL1 status may serve as a potential biomarker for immunotherapy response prediction