L3MBTL2 Antibody

What is the primary biological function of L3MBTL2?

L3MBTL2 serves as a key link between RNF8 and RNF168 in the DNA damage response pathway. Following DNA double-strand breaks (DSBs), L3MBTL2 is recruited by MDC1 to damage sites, then ubiquitylated by RNF8. This ubiquitylated L3MBTL2 subsequently facilitates RNF168 recruitment to DNA lesions, promoting DNA repair . Additionally, L3MBTL2 functions as a transcriptional repressor and is an integral component of atypical polycomb repressive complex 1 (PRC1) that contains E2F6, RING2, HP1γ, and MBLR . In reproductive biology, L3MBTL2 plays crucial roles in chromatin remodeling during both meiosis and spermiogenesis, with its deficiency leading to increased abnormal spermatozoa and progressive decrease in sperm counts .

Where is L3MBTL2 most highly expressed in tissues?

L3MBTL2 is most abundantly expressed in pachytene spermatocytes within the testis . This expression pattern correlates with its functional role in chromatin remodeling during spermatogenesis. Studies using germ cell-specific knockout models have demonstrated that L3MBTL2 deletion in testicular cells leads to progressive testicular failure in aging mice, highlighting the tissue-specific importance of this protein .

What are the key post-translational modifications of L3MBTL2?

L3MBTL2 undergoes two primary post-translational modifications critical to its function:

• Phosphorylation: L3MBTL2 is phosphorylated at ATM/ATR consensus motifs in an ATM-dependent manner following DNA damage. Specifically, S335 has been identified as the critical residue phosphorylated after DNA damage . This modification is essential for proper localization to DNA damage sites.

• Ubiquitylation: Following DNA damage, L3MBTL2 is robustly ubiquitylated in an RNF8-dependent manner . This ubiquitylation occurs at DNA damage sites and is dependent on proper localization, as the S335A phosphorylation-deficient mutant shows reduced ubiquitylation .

What are the recommended applications for L3MBTL2 antibodies?

L3MBTL2 antibodies are versatile tools applicable to several experimental techniques:

These applications have been validated with human and mouse samples, with demonstrated specificity in multiple cell lines including HeLa, U2OS, and MOLT4 .

How should researchers optimize ChIP experiments with L3MBTL2 antibodies?

For optimal ChIP results with L3MBTL2 antibodies:

• Chromatin preparation: Use fresh chromatin extracts from cells with verified L3MBTL2 expression (HeLa cells work well) .

• Antibody amount: Use 5 μg of L3MBTL2 antibody per ChIP reaction .

• Controls: Always include a preimmune rabbit IgG control at equivalent concentration .

• Target verification: The CDC7 gene locus has been validated as a positive control region for L3MBTL2 binding .

• Detection method: PCR with primers targeting the CDC7 gene locus provides reliable detection of precipitated DNA .

What are common pitfalls when using L3MBTL2 antibodies for immunoprecipitation?

When performing immunoprecipitation with L3MBTL2 antibodies, researchers should be aware of:

• Sample quantity: Use sufficient starting material (1000 μg of whole cell lysate is recommended) .

• Antibody specificity: Validate specificity by including controls with preimmune IgG at equivalent concentration (2.5 μg) .

• Western blotting detection: For detecting immunoprecipitated L3MBTL2, use the same antibody at 1:1000 dilution for western blotting .

• Secondary antibody interference: When detecting immunoprecipitated proteins, use anti-rabbit IgG secondary antibodies specifically designed to reduce interference from the IP antibody (e.g., EasyBlot anti-rabbit IgG) .

• Protein-protein interactions: Be aware that L3MBTL2 interacts with other proteins such as RNF8, which may co-immunoprecipitate .

How can researchers study L3MBTL2's role in DNA double-strand break repair?

To investigate L3MBTL2's function in DSB repair:

• DSB induction systems: Use I-SceI-based reporter systems in U2OS cells to induce single DSBs per cell and track L3MBTL2 localization .

• Ionizing radiation: Treat cells with ionizing radiation and assess L3MBTL2 foci formation and co-localization with γH2AX .

• Phosphorylation assessment: Examine ATM-dependent phosphorylation of L3MBTL2 at S335 following DNA damage using phospho-specific antibodies or phosphorylation-deficient mutants (S335A) .

• Ubiquitylation analysis: Investigate RNF8-dependent ubiquitylation of L3MBTL2 before and after DNA damage .

• Downstream effector recruitment: Analyze how L3MBTL2 facilitates RNF168 recruitment to DSBs, potentially using L3MBTL2 knockdown or knockout systems .

How does L3MBTL2 contribute to chromatin remodeling during spermatogenesis?

To study L3MBTL2's role in spermatogenesis:

• Conditional knockout models: Generate germ cell-specific L3MBTL2 knockout models using Cre-loxP systems (e.g., Stra8-icre with floxed L3MBTL2) .

• Meiotic spreads: Prepare meiotic spreads from control and L3MBTL2-knockout testes to examine γH2AX and SYCP3 patterns during different meiotic stages .

• Histone modification analysis: Assess changes in histone modifications (H3K27me3, H3K9me1, H3K4me2) in the XY body and autosomes during meiosis .

• DNA damage marker assessment: Examine γH2AX deposition in leptotene spermatocytes and its retention on autosomes in pachytene spermatocytes .

• RNF8 pathway analysis: Investigate changes in nuclear RNF8 levels, ubH2A levels, histone acetylation in elongating spermatids, and chromatin condensation in sperm .

What experimental approaches can distinguish between L3MBTL2's transcriptional and DNA repair functions?

To differentiate between L3MBTL2's dual roles:

• Domain-specific mutants: Generate mutants that selectively disrupt either L3MBTL2's transcriptional repression domains or its DNA damage response functions (e.g., S335A phosphorylation site mutant) .

• Temporal analysis: Compare immediate responses to DNA damage (minutes to hours) versus long-term transcriptional effects (hours to days).

• ChIP-seq before and after damage: Perform ChIP-seq for L3MBTL2 before and after DNA damage to identify differential binding patterns.

• Protein complex identification: Use mass spectrometry following immunoprecipitation to identify L3MBTL2-associated proteins in undamaged versus damaged conditions.

• RNA-seq with temporal resolution: Conduct RNA-seq analysis in L3MBTL2-deficient versus wild-type cells at different timepoints after DNA damage.

What controls are essential when validating L3MBTL2 antibody specificity?

For rigorous antibody validation:

• Negative controls:

  • Preimmune rabbit IgG at equivalent concentration (5 μg for ChIP, 2.5 μg for IP)

  • L3MBTL2 knockout or knockdown samples

• Positive controls:

  • HeLa or U2OS cell extracts with verified L3MBTL2 expression

  • MOLT4 whole cell lysates for western blot (30 μg loading)

• Target verification:

  • For ChIP: CDC7 gene locus is a verified L3MBTL2 binding site

  • For western blot: Verify molecular weight (expected band around 70-80 kDa)

• Sample preparation:

  • Use 7.5-5% SDS-PAGE for optimal resolution

  • For IP, use 1000 μg whole cell lysate with 2.5 μg antibody

How can researchers optimize the detection of L3MBTL2 in different experimental conditions?

For optimal L3MBTL2 detection:

• Western blot optimization:

  • Use 5-7.5% SDS-PAGE for better resolution of higher molecular weight proteins

  • Try antibody dilutions between 1:500-1:3,000 to determine optimal signal-to-noise ratio

  • Consider enhanced chemiluminescence detection systems for improved sensitivity

• Immunofluorescence optimization:

  • For DNA damage studies, examine L3MBTL2 localization to DNA damage sites using I-SceI-based systems or following ionizing radiation

  • Co-stain with γH2AX to confirm localization to DNA damage foci

• ChIP optimization:

  • For L3MBTL2 ChIP assays, focus on the CDC7 gene locus as a positive control region

  • Use appropriate sonication conditions to generate 200-500 bp chromatin fragments

• Signal amplification:

  • For low-abundance detection, consider using signal amplification methods (e.g., tyramide signal amplification for immunofluorescence)

  • For ChIP-qPCR, optimize primer design for CDC7 and other potential target regions

How should γH2AX and L3MBTL2 co-localization data be interpreted in DNA damage studies?

When analyzing γH2AX and L3MBTL2 co-localization:

• Normal response pattern: L3MBTL2 forms ionizing radiation-induced foci that overlap with γH2AX, indicating successful recruitment to DNA damage sites .

• Temporal dynamics:

  • Initial recruitment requires MDC1

  • Persistence at damage sites depends on ATM-mediated phosphorylation at S335

• Functional significance:

  • Co-localization indicates proper DNA damage response pathway activation

  • Absence of L3MBTL2 at γH2AX foci may suggest defects in the MDC1-RNF8-L3MBTL2-RNF168 pathway

• Quantitative assessment:

  • Measure percentage of γH2AX foci with L3MBTL2 co-localization

  • Analyze intensity correlation between the two markers

What are the implications of L3MBTL2 deficiency on pachytene spermatocyte development?

L3MBTL2 deficiency affects pachytene spermatocytes through several mechanisms:

• Increased initial DNA damage: L3MBTL2-deficient leptotene spermatocytes show increased γH2AX deposition, suggesting higher initial DNA damage load .

• Persistent DNA damage: γH2AX is inappropriately retained on autosomes in pachytene spermatocytes in L3MBTL2-deficient cells , indicating incomplete resolution of DNA damage.

• Progressive deterioration: While young L3MBTL2-deficient mice may show subtle phenotypes, aging mice demonstrate:

  • Increased abnormal spermatozoa

  • Progressive decrease in sperm counts

  • Premature testicular failure

• Chromatin condensation defects: L3MBTL2 deficiency leads to:

  • Decreased levels of RNF8 and ubH2A pathway components

  • Reduced histone acetylation in elongating spermatids

  • Defective protamine 1 deposition

  • Impaired chromatin condensation in sperm

How can researchers determine whether L3MBTL2 phosphorylation affects its ubiquitylation status?

To investigate the relationship between L3MBTL2 phosphorylation and ubiquitylation:

• Phosphorylation-deficient mutants: Compare ubiquitylation levels of wild-type L3MBTL2 versus S335A mutant following DNA damage .

• ATM inhibition: Treat cells with ATM inhibitors before DNA damage induction and assess effects on L3MBTL2 ubiquitylation .

• Sequential analysis: Use time-course experiments to determine whether phosphorylation precedes ubiquitylation following DNA damage.

• Site-specific analysis:

  • Use mass spectrometry to identify specific lysine residues ubiquitylated on L3MBTL2

  • Generate lysine-to-arginine mutants at potential ubiquitylation sites

  • Assess whether phosphorylation at S335 affects ubiquitylation at specific lysine residues

• Domain interaction studies: Investigate whether phosphorylation at S335 creates or disrupts binding interfaces for RNF8 or other E3 ubiquitin ligases.

Research has already established that the S335A phosphorylation-deficient mutant, which fails to localize to DSBs, shows defects in L3MBTL2 ubiquitylation similar to those observed in MDC1-depleted cells , suggesting that proper localization to damage sites is a prerequisite for ubiquitylation.