HSP90B1 Antibody

What is HSP90B1 and what are its alternative names in the literature?

HSP90B1 is an endoplasmic reticulum (ER) chaperone protein that contributes to protein folding in the ER compartment. In the literature, it is also known by several alternative names including:

• gp96

• grp94

• ERp99

• Targ2

• Tra-1

• Tra1

• Hspc4

The gene is officially designated as hsp90b1 (MGI:98817) in mice . It functions alongside another ER chaperone, HSPA5 (also known as Grp78, BiP) (MGI:95835), though they appear to have distinct functions as they do not fully colocalize in cellular studies .

What are the primary applications for HSP90B1 antibodies in research?

HSP90B1 antibodies are utilized in several key research applications:

• Western Blotting: Detects HSP90B1 in cell and tissue lysates at approximately 100 kDa under reducing conditions. This application has been validated in human cell lines (HEK293T, HeLa), mouse cell lines (A20 B cell lymphoma), and rat cell lines (L6 myoblast) .

• Immunohistochemistry: Used for detection of HSP90B1 in paraffin-embedded tissue sections. Specific staining is typically localized to cytoplasm and plasma membrane, as demonstrated in human mesothelioma tissue .

• Knockout Validation Studies: Used to confirm antibody specificity by comparing parental cell lines with HSP90B1 knockout lines .

How can researchers validate HSP90B1 antibody specificity?

Validating HSP90B1 antibody specificity is crucial for reliable research outcomes. Multiple approaches should be employed:

• Knockout Cell Line Comparison: Compare antibody reactivity between parental and HSP90B1 knockout cell lines. As demonstrated with HEK293T human embryonic kidney cell line, a specific band at approximately 100 kDa should be detected in parental cells but absent in knockout cells .

• Loading Controls: Always include appropriate loading controls such as GAPDH to ensure equal protein loading across samples .

• Tissue-Specific Expression: Confirm that expression patterns match known tissue distributions. For example, HSP90B1 should be detectable in B cells but shows differential expression between conventional and innate-like B cells .

• Conditional Knockout Models: In studies using conditional knockout models (e.g., Zp3-cre; Hsp90b1 flox/flox), confirm specific depletion in target tissues while maintaining expression in non-targeted tissues .

What is the expression pattern of HSP90B1 in normal versus cancerous tissues?

HSP90B1 exhibits differential expression between normal and cancerous tissues, with important implications for cancer research:

What role does HSP90B1 play in B-cell biology?

HSP90B1's role in B-cell biology has been clarified through B-cell-specific HSP90B1-null mice studies:

• B-cell Development: Contrary to earlier hypotheses, HSP90B1 is not essential for B-cell development. Knockout B cells develop normally with no apparent problems in plasma cell differentiation, Ig assembly, class-switching, or Ig production .

• TLR Signaling: HSP90B1 is critical for TLR signaling in B cells. Knockout B cells fail to proliferate in response to multiple TLR ligands, resulting in attenuated antibody production in the context of TLR stimulation .

• Integrin Expression: HSP90B1 is required for the expression/function of select integrins, specifically α4 and β2 integrins. Consequently, HSP90B1-null conventional B cells don't accumulate efficiently in lymph nodes, and innate-like B cells fail to compartmentalize properly .

• Germinal Centers: Despite the above defects, HSP90B1 is dispensable for germinal center formation and memory antibody responses in vivo .

What methodological approaches should be used to study HSP90B1 when constitutive knockout is embryonically lethal?

Since constitutive knockout of HSP90B1 causes peri-implantation embryonic lethality, researchers must employ alternative approaches:

• Conditional Knockout Systems: Utilize tissue-specific or temporally controlled Cre-loxP systems. For example, the oocyte-specific conditional knockout line using Zp3-cre with floxed Hsp90b1 (Hsp90b1^flox, MGI:3700023) has been established to study HSP90B1's role in early development .

• Experimental Design Considerations:

  • Confirm knockout efficiency through RT-qPCR and Western blot analysis

  • Compare with appropriate controls (Hsp90b1^flox/flox without Cre)

  • Validate tissue specificity of knockout by immunodetection in target and non-target tissues

  • Consider potential compensatory mechanisms by related chaperones (e.g., HSPA5)

• Developmental Analysis Protocol:

  • For embryonic studies, collect embryos at specific developmental stages

  • Perform morphological and molecular analyses including immunofluorescence for cellular structures

  • Analyze relevant developmental markers to pinpoint the stage of developmental arrest

How can researchers differentiate between HSP90B1 and other ER chaperones like HSPA5 (BiP/GRP78)?

Differentiating between HSP90B1 and other ER chaperones requires careful experimental design:

• Immunofluorescence Co-localization: Despite similar expression profiles, HSP90B1 and HSPA5 do not fully colocalize in cells (particularly in zygotes), suggesting distinct functions. Double immunofluorescence staining with specific antibodies can reveal these differential localization patterns .

• Functional Compensation Analysis: Research shows that even when HSPA5 is overexpressed in Hsp90b1 mutant embryos, it does not compensate for HSP90B1 deficiency, indicating non-redundant functions .

• Client Protein Specificity: HSP90B1 has a more restricted role than previously thought, chaperoning specific client proteins including:

  • Toll-like receptors (TLRs)

  • Select integrins (α4 and β2)

  Unlike earlier hypotheses, HSP90B1 does not appear to chaperone immunoglobulin molecules .

What is the significance of HSP90B1 expression and phosphorylation in cancer prognosis?

HSP90B1 expression and post-translational modifications have important implications for cancer prognosis:

What are the methodological considerations for studying HSP90B1 in early embryonic development?

Studying HSP90B1 in early embryonic development requires specialized approaches:

• Oocyte-Specific Knockout System: Use the Zp3-cre; Hsp90b1^flox/flox system to delete HSP90B1 specifically in growing oocytes while maintaining expression in somatic cells .

• Developmental Milestones Analysis:

  • Zygotic division (ability to reach 2-cell stage)

  • Mitotic spindle formation

  • Cell cycle progression (G2/M transition)

  • Cytoplasmic organization around the zygotic spindle

• Phenotype Characterization:

  • HSP90B1 deficiency in oocytes leads to reduced zona pellucida thickness

  • HSP90B1-null zygotes fail to reach the 2-cell stage

  • Primary defects include G2/M block or abnormal mitotic spindle formation

  • Defective organization of cytoplasmic regions surrounding the zygotic spindle

• Technical Considerations:

  • Carefully stage-match oocytes and embryos for comparative analyses

  • Use appropriate markers for ER compartment and cytoskeleton visualization

  • Consider potential maternal versus zygotic effects in interpretation