LMNB1 Monoclonal Antibody

What is Lamin B1 and what are its main cellular functions?

Lamin B1 is an intermediate filament protein that lines the inner surface of the nuclear envelope. It is encoded by the LMNB1 gene in humans and has a reported amino acid length of 586 with an expected molecular mass of 66.4 kDa . Lamin B1 forms part of the dynamic nuclear lamina structure that is disassembled and reassembled during mitosis.

The protein plays central roles in:

• Chromatin organization and gene positioning

• DNA replication and repair mechanisms

• Cell cycle progression regulation

• Cellular stress responses

• Proliferation and differentiation processes

Research has demonstrated that Lamin B1 loss is associated with cellular senescence and contributes to a broad range of aging-related diseases including cardiovascular diseases and cancers . Its structural role in maintaining nuclear integrity makes it essential for proper cellular function across different tissues.

How does Lamin B1 expression vary across different tissue types?

Lamin B1 shows varied expression patterns across different tissues, which is relevant when designing experimental controls. While Lamin B1 is widely expressed in most cell types, its expression levels can differ significantly.

Based on the search results, Lamin B1 antibodies have shown positive reactions in multiple tissues and cell lines, including:

• Various cancer cell lines: NCI-H1299, HeLa, HepG2, HEK-293, Jurkat, K-562

• Other mammalian cell lines: PC-12, NIH/3T3, 4T1

• Tissue samples: Human pancreas cancer tissue, human breast cancer tissue, mouse eye tissue

Of particular note, research has shown differences in Lamin B1 expression between normal and cancerous tissues. For example, Lamin B1 levels are reduced in lung cancer patients compared to normal lung tissue, while lamin A levels remain unchanged, suggesting distinct functions for different lamin types in lung carcinogenesis .

What is the significance of Lamin B1 farnesylation for nuclear integrity?

Farnesylation of Lamin B1 represents a critical post-translational modification that significantly impacts nuclear morphology and function. Research using genetically modified mouse models (Lmnb1CS/CS) where farnesylation was eliminated has provided important insights into this process.

In cells lacking Lamin B1 farnesylation:

• Nuclear morphology becomes abnormal in approximately 30% of cells

• Lamin B1 distributes in a distinctive "honeycomb" pattern rather than the normal even distribution along the nuclear envelope

• Protein levels of the non-farnesylated Lamin B1 are reduced by approximately 35% compared to wild-type cells

Importantly, studies have confirmed that the abnormal nuclear morphology is primarily attributable to the absence of farnesylation rather than simply reduced protein levels. Comparisons between Lmnb1CS/CS and Lmnb1+/− cells, which have similar reductions in Lamin B1 levels, showed that honeycomb nuclear morphology was significantly more frequent in cells lacking farnesylation (29.9% vs. 14.3%) .

These findings demonstrate that farnesylation is essential for proper retention of Lamin B1 in the nuclear envelope and maintenance of nuclear integrity.

How does Lamin B1 contribute to the regulation of somatic hypermutation in B cells?

Lamin B1 functions as a negative epigenetic regulator of somatic hypermutation (SHM) in B cells, effectively serving as a "mutational gatekeeper" that suppresses aberrant mutations potentially driving lymphoid malignancy . This regulatory role involves nuclear architecture and chromatin organization.

Research has revealed several key aspects of this mechanism:

• During B-cell activation and formation of lymphoid germinal centers, genome binding of Lamin B1 is reduced

• ChIP-Seq analysis demonstrated that kappa and heavy variable immunoglobulin domains are released from the Lamin B1 suppressive environment when SHM is induced in B cells

• RNA interference-mediated reduction of Lamin B1 resulted in spontaneous SHM as well as kappa-light chain aberrant surface expression

These findings establish Lamin B1 as a critical component in maintaining genomic stability in B cells. When Lamin B1 binding is reduced, certain genomic regions become more accessible to the molecular machinery responsible for introducing mutations during SHM, an essential process for antibody diversification in the adaptive immune response .

What is the relationship between Lamin B1 expression and cancer progression?

Lamin B1 expression patterns have significant implications for cancer progression and patient outcomes. Research has identified both decreased and increased expression depending on the cancer type, suggesting context-dependent roles.

In lung cancer:

• Lamin B1 levels are reduced compared to normal lung tissue

• Lower expression of Lamin B1 is associated with higher tumor grade

• Lamin B1 deficiency has been shown to promote lung cancer development and metastasis through epigenetic derepression of RET

In B-cell malignancies:

In colon and pancreatic cancers:

• Counterintuitively, overexpression of Lamin B1 can indicate poor prognosis

These varying patterns highlight the complex and tissue-specific roles of Lamin B1 in cancer biology, suggesting that it may function differently depending on cellular context and cancer type.

How does Lamin B1 deficiency affect neuronal development and brain structure?

Research using genetic models has revealed crucial roles for Lamin B1 in neuronal development and brain morphogenesis. Studies with Lmnb1CS/CS mice (lacking Lamin B1 farnesylation) and Lmnb1−/− mice (completely lacking Lamin B1) demonstrate significant developmental abnormalities.

In Lmnb1CS/CS mice:

• Newborns exhibit a flattened cranium and smaller brain size compared to wild-type mice

• The midbrain is most prominently affected (21.5 ± 7.6% smaller than in wild-type mice), with the cortex also reduced (9.1 ± 4.6% smaller)

• Abnormal layering of cortical neurons is observed, though milder than in Lmnb1−/− embryos

• Many neurons in the midbrain are markedly elongated with naked chromatin devoid of surrounding lamina

A striking cellular phenotype involves "dumbbell-shaped" nuclei where:

• The lamin B1-containing end of the nucleus positions in the leading edge of migrating neurons

• The "naked chromatin" without lamina positions in the trailing edge

• Both ends remain connected by a thin strand of DNA

These abnormalities help explain why Lmnb1CS/CS mice die soon after birth, similar to complete knockout mice, and highlight the essential nature of properly farnesylated Lamin B1 for normal brain development and neuronal migration .

What are the optimal conditions for using LMNB1 monoclonal antibodies in different applications?

The optimal conditions for LMNB1 monoclonal antibody use vary by application. Based on validated protocols, the following dilutions and conditions are recommended:

These conditions have been validated across multiple cell types including NCI-H1299, HeLa, HepG2, HEK-293, Jurkat, K-562, PC-12, NIH/3T3, and 4T1 cells .

For research involving somatic hypermutation studies, protocols have successfully used LMNB1 siRNA transfection followed by induction of SHM, with DNA isolation 72 hours after initial treatment .

How should nuclear morphology be assessed when studying Lamin B1 functions?

When studying Lamin B1 functions, particularly in the context of nuclear morphology, several validated approaches should be considered:

• Immunofluorescence microscopy with co-staining:

  • Lamin B1 antibody staining to visualize lamina distribution

  • DNA markers (DAPI, Hoechst) to visualize chromatin

  • Additional nuclear envelope markers such as LAP2β (lamin-associated polypeptide 2) to visualize the nuclear membrane

  • Pericentrin or other cytoskeletal markers to establish cell polarity

• Quantitative assessment of nuclear abnormalities:

  • Score the frequency of abnormal nuclear morphologies (e.g., honeycomb pattern, dumbbell-shaped nuclei)

  • Compare between experimental conditions (e.g., Lmnb1CS/CS vs. Lmnb1+/− vs. wild-type)

  • Consider statistical analysis to determine significance of observed differences

• Combined with functional assays:

  • For neuronal studies, assess migration patterns in conjunction with nuclear morphology

  • For B-cell studies, measure somatic hypermutation rates or kappa-light chain expression

This multi-parameter approach provides a comprehensive understanding of how Lamin B1 alterations affect nuclear structure and downstream cellular functions.

What controls should be included when analyzing Lamin B1 expression in cancer studies?

When analyzing Lamin B1 expression in cancer studies, appropriate controls are critical for meaningful interpretation. Based on research practices, the following controls should be considered:

• Tissue-matched normal controls:

  • Include normal tissue from the same organ to establish baseline expression

  • For lung cancer studies, normal lung tissue serves as an essential comparison point

  • Consider matched adjacent normal tissue when possible

• Other lamin protein controls:

  • Measure lamin A/C expression as internal controls

  • Research shows that while Lamin B1 levels may be altered in cancers, lamin A levels might remain unchanged, highlighting the specificity of Lamin B1 alterations

• Genetic manipulation controls:

  • When using siRNA to reduce Lamin B1 expression, include scrambled siRNA controls

  • For studies examining correlations with mutations, include wild-type controls alongside mutant samples

• Positive tissue controls for antibody validation:

  • Include known high-expressing tissues such as colon, breast, fallopian tube, tonsil, testis when validating antibodies

  • Use well-characterized cancer cell lines with established Lamin B1 expression patterns

• For prognostic studies:

  • Include stratification by established prognostic factors to isolate the specific contribution of Lamin B1 expression

  • For CLL studies, controls should account for other known prognostic factors to establish independent significance

These controls help distinguish cancer-specific alterations from technical variability or tissue-specific expression patterns.

What are common issues with detecting Lamin B1 in immunofluorescence studies and how can they be resolved?

Researchers may encounter several challenges when detecting Lamin B1 using immunofluorescence. Here are common issues and their solutions:

• Honeycomb pattern misinterpretation:

  • Issue: A honeycomb distribution of Lamin B1 might be interpreted as a technical artifact rather than a biological phenotype.

  • Solution: Compare with known controls like Lmnb1CS/CS cells where this pattern is expected (29.9 ± 13.6% of cells). The honeycomb pattern represents a biological phenotype in cells with non-farnesylated Lamin B1 .

• Antigen accessibility problems:

  • Issue: Nuclear envelope proteins may be difficult to access due to chromatin compaction or fixation issues.

  • Solution: Optimize antigen retrieval with TE buffer pH 9.0 or alternatively citrate buffer pH 6.0. Proper fixation and permeabilization protocols are critical for nuclear envelope proteins .

• Signal intensity variations:

  • Issue: Lamin B1 expression varies between tissues and cell types, leading to inconsistent signal intensity.

  • Solution: Adjust antibody concentration based on the specific tissue or cell type (1:200-1:800 for paraffin sections, 1:250-1:1000 for cell culture) . Include positive control tissues such as colon, breast, or testis where Lamin B1 expression is well-characterized.

• Co-localization assessment difficulties:

  • Issue: Determining spatial relationships between Lamin B1 and other nuclear structures.

  • Solution: Implement multi-color immunofluorescence with established markers like LAP2β for nuclear membrane, pericentrin for centrosomes, and DNA stains. This approach was successfully used to characterize the unique "dumbbell-shaped" nuclei in Lmnb1CS/CS neurons .

These troubleshooting strategies can significantly improve the reliability and interpretability of Lamin B1 immunofluorescence studies.

How can researchers accurately quantify changes in Lamin B1 expression across experimental conditions?

Accurate quantification of Lamin B1 expression changes requires systematic approaches that account for technical and biological variability:

• Western blot quantification:

  • Use appropriate loading controls (housekeeping proteins)

  • Implement densitometry analysis with normalization

  • Compare relative expression levels (e.g., research showed lamin B1 levels in Lmnb1CS/CS MEFs were ∼35% lower than in wild-type MEFs)

  • Multiple biological replicates are essential (at least 3)

• qRT-PCR for transcript quantification:

  • Particularly useful to differentiate between transcriptional and post-transcriptional effects

  • Research showed normal Lmnb1 transcript levels in Lmnb1CS/CS mice despite reduced protein levels, indicating post-transcriptional regulation

  • Use validated reference genes for normalization

• Immunofluorescence quantification approaches:

  • Measure nuclear rim intensity with line scans across nuclei

  • Quantify frequency of abnormal nuclear morphologies (honeycomb pattern)

  • Implement automated image analysis for unbiased assessment of large cell populations

  • Score based on established criteria (e.g., research demonstrated correlation between non-farnesylated lamin B1 levels and frequency of honeycomb nuclei)

• Flow cytometry for high-throughput analysis:

  • Provides quantitative assessment of protein levels across large cell populations

  • Allows concurrent analysis of cell cycle phases or other parameters

  • Validated for intracellular staining of Lamin B1 in HeLa cells

By combining multiple quantification approaches, researchers can obtain robust and reproducible measurements of Lamin B1 expression changes across experimental conditions.

What are the key considerations when using LMNB1 antibodies for studying B-cell somatic hypermutation?

When studying B-cell somatic hypermutation (SHM) in relation to Lamin B1, researchers should consider several critical factors:

• Temporal dynamics of experimental design:

  • For combined antibody/siRNA treatments, follow established protocols: induce SHM 48 hours after LMNB1 siRNA transfection, then isolate DNA 72 hours after initial treatment

  • This timing allows for effective protein knockdown before initiating the hypermutation process

• Appropriate controls for mutation analysis:

  • Include wild-type cells alongside Lamin B1-depleted cells

  • Use scrambled siRNA controls to account for non-specific effects of transfection

  • Consider alternative approaches to Lamin B1 depletion (CRISPR, shRNA) for validation

• Comprehensive mutation assessment:

  • Analyze both spontaneous and induced mutations

  • Examine mutations in immunoglobulin variable regions and other genomic loci to determine specificity

  • Investigate kappa-light chain aberrant surface expression, which has been observed following Lamin B1 reduction

• Chromatin organization analysis:

  • Combine with Chromatin Immunoprecipitation-Seq (ChIP-Seq) analysis to examine how Lamin B1 binding patterns change during B-cell activation

  • Research has shown that kappa and heavy variable immunoglobulin domains are released from the Lamin B1 suppressive environment during SHM induction

  • Consider analysis of Lamina-Associated Domains (LADs) and their relationship to mutational patterns

These considerations will help ensure robust and reproducible results when investigating Lamin B1's role in regulating somatic mutations in B-cells.