RNF26 Antibody

What is RNF26 and what cellular functions does it mediate?

RNF26 is an ER-embedded RING finger protein that functions as an E3 ubiquitin ligase. It plays a critical role in spatiotemporal compartmentalization of the endoplasmic reticulum (ER) by binding to perinuclear vimentin intermediate filaments. This interaction helps integrate ER function with cytoskeletal organization . RNF26 has been demonstrated to regulate membrane dynamics, with its depletion resulting in enhanced motility of the perinuclear ER segment . Beyond structural roles, RNF26 has been implicated in cancer progression through cell cycle regulation, particularly in bladder cancer via the FOXM1/RNF26/p57 axis .

What is the structural organization of RNF26 protein?

RNF26 contains a C-terminal RING domain (amino acids 304-433) that is crucial for its E3 ligase activity and protein interactions. This domain can directly bind to vimentin intermediate filaments . The protein also contains transmembrane segments that anchor it to the ER membrane, with its RING domain extending into the cytoplasm where it can interact with cytoskeletal elements and ubiquitylation substrates . Commercial antibodies are available against various regions, including the central region (amino acids 247-273) and other epitopes recognized by monoclonal antibodies .

How does RNF26 interact with the cytoskeleton?

RNF26 directly binds to vimentin intermediate filaments through its C-terminal RING domain. This interaction is enhanced when RNF26 is catalytically inactive, such as with the I382R mutation . In cells, RNF26 shows partial colocalization with vimentin structures, and this colocalization increases significantly with catalytically inactive RNF26-I382R mutants . This direct binding was confirmed through experiments showing that purified fluorescently-labeled RING domain (Rhodamine-RING-I382R) can be precipitated by recombinant His-tagged vimentin .

What types of RNF26 antibodies are available for research applications?

Multiple types of RNF26 antibodies are commercially available, including:

Researchers should select antibodies based on their specific experimental requirements, considering factors such as clonality, target epitope, and validated applications .

What methods can be used to validate RNF26 antibody specificity?

To ensure specificity of RNF26 antibodies, researchers should:

• Compare staining patterns in wild-type cells versus RNF26 knockout or knockdown cells

• Perform peptide competition assays using the immunizing peptide

• Verify molecular weight specificity through Western blot analysis (expected RNF26 band should be detected)

• For immunofluorescence applications, compare staining patterns with published localization data showing characteristic perinuclear ER distribution

• Use positive control samples with known RNF26 expression, such as Y79 cell line lysates which have been validated for some commercial antibodies

What are the optimal conditions for Western blot detection of RNF26?

For optimal Western blot detection of RNF26:

• Use 35-50 μg of total protein lysate per lane to ensure sufficient detection of endogenous RNF26

• For commercial monoclonal antibodies, a concentration of 1-5 μg/mL is typically recommended

• Include appropriate positive control lysates, such as Y79 cell line lysates, which have been validated with commercial antibodies

• Consider membrane transfer conditions carefully, as RNF26 contains transmembrane domains that may affect transfer efficiency

• When studying RNF26 mutants or fragments, ensure your antibody's epitope is preserved in the construct of interest

How can RNF26 antibodies be used to study its interaction with vimentin?

To investigate RNF26-vimentin interactions:

• Co-immunoprecipitation using RNF26 antibodies can help identify interaction partners, though direct protein-protein interaction studies require careful buffer optimization due to RNF26's membrane localization

• Proximity ligation assays can detect in situ interactions between RNF26 and vimentin

• Immunofluorescence co-localization analysis can reveal spatial relationships between RNF26 and vimentin filaments, particularly when examining catalytically inactive RNF26-I382R which shows enhanced filamentous distribution aligned with vimentin fibers

• In vitro binding assays using purified components can establish direct binding, as demonstrated with Rhodamine-labeled RING domain fragments and His-tagged vimentin

• Biotinylation proximity assays using RNF26 fused to TurboID can identify in vivo proximity partners

What experimental approaches can reveal RNF26's role in ER compartmentalization?

To study RNF26's function in ER organization:

• Immunofluorescence microscopy comparing wild-type, RNF26-depleted, and vimentin knockout cells can reveal alterations in ER morphology and distribution

• Live-cell imaging using ER markers in RNF26-depleted cells can demonstrate enhanced motility of perinuclear ER segments

• ER stress experiments using Tunicamycin treatment can reveal RNF26's role in ERQC (ER quality control) compartment formation, which depends on both RNF26 and vimentin

• Co-staining for RNF26 and ER markers like VAP-A can quantify the degree of perinuclear ER retention mediated by RNF26

• Rescue experiments reintroducing wild-type or mutant RNF26 into knockout cells can establish structure-function relationships

How does catalytic activity affect RNF26 localization and function?

RNF26's catalytic activity significantly influences its localization and interaction partners:

• Catalytically inactive RNF26 (I382R mutant) shows enhanced binding to vimentin compared to wild-type RNF26

• While RNF26 interactions with endosomal adaptors (TOLLIP, EPS15, TAX1BP1) depend on its E3 ligase activity, vimentin binding is enhanced by catalytic inactivation

• Wild-type RNF26 shows partial colocalization with vimentin, while the I382R mutant displays pronounced filamentous distribution aligned with vimentin fibers

• In vimentin knockout cells, catalytically inactive RNF26 loses its perinuclear filamentous distribution and forms aggregate-like clusters, suggesting vimentin dependency for proper localization

• The presence of specific E2 enzymes (UBE2J1 vs. UBE2J2) influences RNF26 activity states and may dictate preferential binding to cytoskeleton versus endolysosomal components

What is known about RNF26's role in cancer progression?

RNF26 has been implicated in cancer progression, particularly in bladder cancer:

• Bioinformatics analyses have identified RNF26 as a risk factor for bladder cancer

• RNF26 is abnormally upregulated in bladder cancer cells and tissues, with higher expression correlating with unfavorable prognosis

• Mechanistically, RNF26 expression is promoted by FOXM1 at the transcriptional level through the MuvB complex

• Upregulated RNF26 degrades p57 (CDKN1C), a cell cycle regulator, thereby promoting cell cycle progression and cancer development

• The FOXM1/RNF26/p57 signaling axis represents a potential therapeutic target for bladder cancer treatment

How can RNF26 antibodies be used to study ER stress responses?

RNF26 plays important roles in ER stress responses and can be studied using antibodies:

• Western blot analysis can detect RNF26 upregulation in parallel with HERP1 following Tunicamycin-induced ER stress

• Immunofluorescence microscopy can visualize the formation of ERQC (ER Quality Control) compartments during stress, which depends on both RNF26 and vimentin

• Co-staining for RNF26 and endolysosomal markers (CD63) can reveal perinuclear clustering of endolysosomes during ER stress, which requires RNF26

• Antibodies against RNF26 and its binding partners (UBE2J1, HERP1/2) can help identify how these protein complexes assemble during stress responses

• Time-course experiments with synchronized stress induction can reveal the temporal dynamics of RNF26 involvement in stress pathway activation

What are common challenges when using RNF26 antibodies?

Researchers may encounter several challenges when working with RNF26 antibodies:

• As a membrane protein, RNF26 may require specialized extraction buffers containing appropriate detergents for efficient solubilization

• Cross-reactivity with other RING domain-containing proteins may occur, necessitating careful validation

• When studying overexpressed RNF26, high expression levels may alter normal localization patterns or induce aggregation, particularly with catalytically inactive mutants

• For immunofluorescence, maintaining intact ER and cytoskeletal structures during fixation is crucial for accurate localization studies

• When detecting RNF26-mediated ubiquitylation events, deubiquitinase inhibitors may be necessary to preserve modification status

What controls are essential for RNF26 antibody experiments?

Critical controls for RNF26 antibody experiments include:

• RNF26 knockout or knockdown cells as negative controls

• Rescue experiments with wild-type RNF26 to confirm specificity of observed phenotypes

• Catalytically inactive RNF26 (I382R) as a control for ubiquitin ligase activity-dependent functions

• Secondary antibody-only controls to assess background staining

• For co-localization studies, appropriate markers for ER (VAP-A), vimentin, and endolysosomal compartments (CD63)

• When studying protein interactions, catalytically inactive RNF26 may yield stronger signals due to enhanced binding to partners like vimentin