FKBP11 Antibody

What is FKBP11 and why is it significant in immunological research?

FKBP11 (FK506-binding protein 11), also known as FKBP19, is a 201 amino acid single-pass membrane protein belonging to the FKBP-type peptidyl-prolyl isomerase (PPIase) family. It catalyzes the folding of proline-containing polypeptides and is primarily expressed in secretory tissues including pancreas, pituitary, stomach, lymph nodes, and salivary glands . Recent research has identified FKBP11 as a plasma cell-specific protein with elevated expression in conditions such as idiopathic pulmonary fibrosis (IPF) . Its significance lies in its role as a novel antibody folding catalyst regulated by X-box-binding protein 1 (XBP1) as part of the unfolded protein response (UPR) . Understanding FKBP11 function provides insights into plasma cell biology, antibody production mechanisms, and potential therapeutic targets for autoimmune diseases.

How does FKBP11 differ from other members of the FKBP family?

FKBP11 contains one PPIase FKBP-type domain and is encoded by a gene that maps to human chromosome 12q13.12 . Unlike several other FKBP family members, FKBP11 is specifically localized to antibody-producing plasma cells . It is primarily expressed in the endoplasmic reticulum and shows high expression in immune-related tissues such as the spleen and lymph nodes . FKBP11 is particularly distinguished by its induction during B cell to plasma cell differentiation and its role in the plasma cell unfolded protein response . While many FKBPs are broadly expressed, FKBP11's restricted expression pattern in secretory tissues and plasma cells suggests specialized functions in protein folding within these specific cellular contexts. Like other FKBPs, its PPIase activity is inhibited by FK506 (tacrolimus) and rapamycin .

What are the known physiological functions of FKBP11?

FKBP11 functions as an antibody peptidyl-prolyl cis-trans isomerase that assists in the proper folding of immunoglobulins . Research has demonstrated that recombinant human FKBP11 can refold IgG antibody in vitro, a function inhibited by FK506 . FKBP11 is induced as part of the unfolded protein response (UPR) in an XBP1-dependent manner, suggesting its importance in managing endoplasmic reticulum stress in plasma cells . Additionally, FKBP11 appears to have protective functions against ER stress-mediated cell death, as FKBP11 deficiency increases susceptibility to such stress in alveolar epithelial cells . Beyond its role in antibody folding, recent research has implicated FKBP11 in cell proliferation, migration, and invasion in certain cancer contexts, particularly clear cell renal cell carcinoma (ccRCC) .

What are the optimal conditions for using FKBP11 antibody in Western blotting?

When using FKBP11 antibody for Western blotting, optimal dilution ratios typically range from 1:100 to 1:1000 based on manufacturer specifications . Sample preparation should include proper protein extraction methods that preserve endoplasmic reticulum proteins. For tissues with high FKBP11 expression (lymphoid tissues, secretory organs), lower antibody concentrations may suffice, while tissues with lower expression may require higher concentrations. Blocking should be performed with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature. Primary antibody incubation is recommended overnight at 4°C, followed by appropriate secondary antibody incubation (typically anti-rabbit IgG if using rabbit polyclonal antibodies like those described in the sources) . To validate specificity, positive controls from plasma cells or lymphoid tissues are recommended, while negative controls should include non-secretory tissues or experiments using FKBP11 knockdown samples.

How can researchers optimize immunohistochemical detection of FKBP11 in tissue samples?

For optimal immunohistochemical detection of FKBP11, researchers should use dilutions in the range of 1:100-500 for paraffin-embedded tissues (IHC-P) . Antigen retrieval is critical due to the endoplasmic reticulum localization of FKBP11; citrate buffer (pH 6.0) heat-induced epitope retrieval is generally effective. When investigating FKBP11 in pathological contexts, such as idiopathic pulmonary fibrosis (IPF), co-staining with plasma cell markers (e.g., CD138) is recommended to confirm the cell type-specific expression pattern . Tissue processing should minimize autolysis time to preserve antigenicity. For maximum sensitivity in detecting FKBP11 in plasma cells, amplification systems such as polymer-based detection methods may be beneficial. Counterstaining should be optimized to provide cellular context without obscuring specific signals, with hematoxylin dilution and timing adjusted according to tissue type and thickness.

What experimental approaches can be used to study FKBP11 function in plasma cells?

To study FKBP11 function in plasma cells, researchers can employ several methodological approaches:

• B cell differentiation models: In vitro B cell to plasma cell differentiation models can be used to track FKBP11 induction, as plasma cell-specific FKBP11 expression accompanies this differentiation process .

• RNA interference: siRNA-mediated knockdown of FKBP11 in antibody-producing cell lines (e.g., hybridoma cells) can assess its role in antibody production and secretion. Electroporation has been successfully used for transfection at settings of 950 μF capacity and 300 V .

• Recombinant protein studies: Purified recombinant FKBP11 can be used in in vitro antibody refolding assays. Typical concentrations for refolding experiments are around 0.66 μg/mL of antibody .

• Inhibitor studies: FK506 at approximately 180 μM can be used to inhibit FKBP11's PPIase activity in biochemical assays .

• ER stress induction: Agents inducing ER stress can be used to study FKBP11 upregulation through the unfolded protein response pathway and its XBP1 dependence .

• In vivo models: Analysis of FKBP11 expression in disease models characterized by plasma cell involvement, such as autoimmune conditions or antibody-mediated disorders.

How does FKBP11 expression correlate with pathological conditions?

FKBP11 expression has been found to be significantly altered in several pathological conditions. In idiopathic pulmonary fibrosis (IPF), FKBP11 shows elevated expression (Fold Change of +2.2) specifically in antibody-producing plasma cells within IPF lungs . This correlates with observations of higher levels of circulating plasmablasts and autoantibodies towards lung antigens in IPF patients . In clear cell renal cell carcinoma (ccRCC), FKBP11 expression is also increased, potentially contributing to cancer progression as knockdown of FKBP11 has been shown to inhibit proliferation, migration, and invasion of ccRCC cell lines . Interestingly, the mechanism for FKBP11 upregulation in ccRCC appears to involve epigenetic alterations rather than genomic changes, as FKBP11 shows a low degree of methylation in tumor tissues . These findings suggest that FKBP11 expression profiling could potentially serve as a biomarker for specific pathological states and might represent a therapeutic target.

What is the relationship between FKBP11 and the unfolded protein response (UPR) in plasma cells?

FKBP11 is intricately linked to the unfolded protein response (UPR) in plasma cells through its regulation by X-box-binding protein 1 (XBP1) . Research has demonstrated that FKBP11 expression is induced in the context of ER stress as part of the UPR. The relationship follows this mechanistic pathway:

• ER stress triggers the UPR, activating IRE1α (Inositol-requiring enzyme 1α)

• Activated IRE1α mediates unconventional splicing of XBP1 mRNA

• Spliced XBP1 (XBP1s) acts as a transcription factor

• XBP1s upregulates FKBP11 expression as part of the plasma cell UPR program

This XBP1-dependent regulation is particularly significant in plasma cells, which experience substantial ER stress due to their high rate of antibody production. The induction of FKBP11 appears to be a specialized adaptation that helps plasma cells manage the folding requirements of immunoglobulin production . Despite its apparent importance in the UPR, knockdown studies in hybridoma cells have shown that FKBP11 depletion alone does not dramatically affect antibody secretion, suggesting functional redundancy with other protein folding machinery components in the ER .

What are the potential therapeutic implications of targeting FKBP11 in autoimmune diseases?

The discovery of FKBP11 as a plasma cell-specific protein involved in antibody folding opens several therapeutic possibilities for autoimmune diseases where pathogenic autoantibodies play a crucial role . Unlike current B cell-depleting therapies (e.g., rituximab targeting CD20) that show limited impact on established plasma cell populations, targeting FKBP11 could potentially affect antibody production by long-lived plasma cells. Therapeutic approaches could include:

• Small molecule inhibitors: Development of specific inhibitors targeting FKBP11's PPIase activity, building on the known inhibition by FK506 but with greater specificity to reduce systemic immunosuppressive effects .

• XBP1-FKBP11 pathway modulation: Targeting the upstream regulator XBP1 to indirectly reduce FKBP11 expression and potentially impact plasma cell function during autoimmune flares.

• Antibody folding disruption: Selective inhibition of FKBP11 in autoantibody-producing plasma cells could potentially reduce autoantibody production without broadly suppressing protective immunity.

• Combined approaches: Using FKBP11 inhibition in combination with other therapies, such as extracellular cleavage of autoantibodies, which has proven successful in murine models of autoimmune disease .

How can researchers address potential cross-reactivity of FKBP11 antibodies with other FKBP family members?

Addressing cross-reactivity concerns with FKBP11 antibodies requires multiple validation approaches:

• Epitope selection: Choose antibodies raised against regions unique to FKBP11, avoiding conserved PPIase domains shared with other FKBP family members .

• Specificity verification: Validate antibody specificity using FKBP11 knockdown or knockout samples as negative controls .

• Expression pattern validation: Confirm that the detected expression pattern matches FKBP11's known localization in secretory tissues and plasma cells .

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of FKBP11 and compare their detection patterns.

• Western blot molecular weight verification: Confirm that detected bands correspond to FKBP11's expected molecular weight (approximately 19 kDa) .

• Pre-absorption controls: Perform pre-absorption tests with recombinant FKBP11 to confirm specificity.

• Cross-validation with other techniques: Corroborate immunodetection results with RNA expression data (qRT-PCR, RNA-seq) to confirm that protein detection correlates with mRNA expression patterns .

What factors affect the reproducibility of FKBP11 antibody-based experiments in different tissue types?

Several factors can influence the reproducibility of FKBP11 antibody-based experiments across different tissue types:

• Tissue-specific expression levels: FKBP11 shows variable expression across tissues, with higher levels in secretory tissues (pancreas, pituitary, stomach, lymph nodes, salivary glands) and plasma cells . This variability necessitates optimization of antibody dilutions for each tissue type.

• Fixation and processing methods: Overfixation can mask epitopes, particularly for ER-resident proteins like FKBP11. Fixation duration and type should be standardized across experiments.

• Antigen retrieval efficiency: Different tissues may require optimized antigen retrieval methods due to varying protein crosslinking during fixation .

• Endogenous peroxidase activity: Tissues with high peroxidase activity (e.g., blood-rich tissues) may require more thorough blocking to prevent background in IHC.

• Plasma cell density: Since FKBP11 is highly expressed in plasma cells, tissues with varying plasma cell infiltration (e.g., inflammatory conditions) will show different detection patterns .

• ER stress status: As FKBP11 is upregulated during ER stress, the physiological or pathological state of the tissue can affect expression levels .

• Sample handling and storage: Degradation of FKBP11 during sample processing can vary between tissue types, affecting detection sensitivity.

To maximize reproducibility, researchers should develop tissue-specific protocols with optimized antibody concentrations, incubation times, and detection methods.

How can the functionality of FKBP11 antibodies be validated for specific research applications?

Validating FKBP11 antibody functionality for specific research applications requires application-specific approaches:

• For Western blotting:

  • Verify detection of a band at the correct molecular weight (approximately 19 kDa)

  • Include positive controls (lymphoid tissue extracts) and negative controls (FKBP11 knockdown samples)

  • Confirm signal reduction after FKBP11 siRNA treatment

  • Test different extraction methods to optimize FKBP11 recovery from ER membranes

• For immunohistochemistry (IHC):

  • Validate tissue expression patterns against known FKBP11 distribution

  • Perform co-localization studies with ER markers and plasma cell markers (e.g., CD138)

  • Compare normal vs. pathological tissues (e.g., IPF lungs) to confirm expected expression differences

  • Test multiple fixation and antigen retrieval protocols

• For immunofluorescence:

  • Optimize fixation to preserve ER structure

  • Co-stain with established ER markers

  • Use recommended dilutions (1:50-200 for IF-IHC-P)

  • Include appropriate fluorophore-matched controls

• For functional assays:

  • Validate antibody inhibition of FKBP11's PPIase activity in vitro

  • Compare effects with known inhibitors like FK506

  • Establish dose-response relationships for inhibitory antibodies

• For immunoprecipitation:

  • Confirm pull-down of FKBP11 by mass spectrometry

  • Verify co-immunoprecipitation of known interacting partners

For each application, antibody performance should be benchmarked against established experimental systems where FKBP11 function has been characterized.

Optimal Experimental Parameters for FKBP11 Antibody Applications

FKBP11 Expression Across Different Tissue and Cell Types

Comparative Analysis of FKBP Family Members in Research Applications