ZNF423 Antibody

What are the primary applications of ZNF423 antibodies in mechanistic studies?

ZNF423 antibodies are predominantly used in Western blot (WB), immunohistochemistry (IHC), and chromatin immunoprecipitation (ChIP). For WB, lysates from tissues/cells expressing ZNF423 (e.g., brain, heart, or neuroblastoma cell lines) are recommended, with validation via siRNA knockdown to confirm band specificity at ~144 kDa . In IHC, antigen retrieval protocols using citrate buffer (pH 6.0) improve epitope detection in nuclear compartments . ChIP-grade antibodies require crosslinking optimization (e.g., 1% formaldehyde for 10 min) and validation using positive controls like SMAD1/5-binding regions in BMP-responsive genes .

Table 1: Common ZNF423 Antibody Clones and Applications

How do researchers address antibody specificity challenges for ZNF423?

Specificity validation involves three complementary approaches:

• Genetic knockout controls: CRISPR-Cas9-mediated ablation of ZNF423 in cell lines (e.g., SEM leukemia cells) confirms loss of signal in WB/IHC .

• Isoform discrimination: ZNF423 has two major isoforms (α and β). Antibodies targeting the C-terminal region (e.g., ab216970) detect both isoforms, while N-terminal-specific antibodies distinguish them .

• Cross-reactivity screens: Testing across species (e.g., mouse, rat) ensures antibodies do not bind paralogs like ZNF521 .

What cellular models are optimal for studying ZNF423 function?

• Neuroblastoma: SH-SY5Y cells treated with retinoic acid (RA) to study ZNF423’s role in differentiation .

• Leukemia: SEM cells (KMT2A-AFF1 rearranged) for BMP2-induced SMAD1/5 signaling and ZNF423-EBF1 interactions .

• Embryonic models: F9 teratocarcinoma cells for RA-responsive reporter assays (e.g., RARE-Luc) .

How does ZNF423 mediate context-dependent transcriptional activation vs. repression?

ZNF423’s dual functionality arises from its interaction partners:

• Activation: In BMP signaling, ZNF423 binds SMAD1/5/9 to enhance transcription of BMP target genes (e.g., ID1). Co-IP assays with anti-SMAD1 antibodies and luciferase reporters under BMP-responsive promoters are used to quantify this activity .

• Repression: ZNF423 inhibits EBF1-driven transcription in B-cell development by binding EBF1’s DNA-binding domain, blocking access to target sites like CD79A. FRET assays and ChIP-seq in ZNF423-overexpressing cells demonstrate reduced EBF1 occupancy .

Table 2: Context-Dependent ZNF423 Interactions

What experimental strategies resolve contradictions in ZNF423’s role across cancer types?

In neuroblastoma, high ZNF423 correlates with RA sensitivity and favorable prognosis, whereas in B-cell acute lymphoblastic leukemia (ALL), overexpression predicts poor survival . To reconcile this:

• Pathway-specific inhibition: Use small molecules (e.g., LDN-193189 for BMP inhibition) to isolate ZNF423’s BMP-dependent vs. RA-dependent effects .

• Single-cell RNA-seq: Resolve heterogeneous ZNF423 expression within tumor subpopulations .

• In vivo xenografts: Compare tumor growth in ZNF423-knockout vs. wild-type models under RA/BMP2 treatment .

How do post-translational modifications (PTMs) regulate ZNF423 activity?

Phosphoproteomics studies identify three critical PTMs:

• S743 phosphorylation (by CDK2): Enhances ZNF423-SMAD1 binding in BMP signaling .

• Ubiquitination at K892: Promotes proteasomal degradation; blocked by BMP2 via SMURF1 inhibition .

• Acetylation at K45: Impairs EBF1 binding, assessed via co-IP with anti-acetyllysine antibodies .

Methodological Recommendations

• Co-IP buffers: Use RIPA buffer with 300 mM NaCl to disrupt weak ZNF423-EBF1 interactions .

• ChIP-seq peak calling: Combine MACS2 with manual curation to filter non-specific zinc finger binding sites .

• Statistical thresholds: For RNA-seq, apply |log2FC| > 1.5 and FDR < 0.01 to identify ZNF423-regulated genes .