tmem55ba Antibody

What is TMEM55B and what cellular functions does it regulate?

TMEM55B (transmembrane protein 55B) is a lysosomal protein that plays crucial roles in cellular homeostasis during oxidative stress. Recent research has identified that TMEM55B contributes to cellular stress responses through three distinct mechanisms. First, it facilitates the recruitment of HECT domain E3 ligases like NEDD4 to lysosomal surfaces via a highly conserved PPXY motif. This interaction enables the ubiquitination and subsequent proteasomal degradation of PLEKHM1, which halts the fusion of autophagosomes with potentially damaged lysosomes . Second, TMEM55B interacts with components of the ESCRT complex through a PSAP motif to facilitate lysosomal repair mechanisms. Third, it has been shown to sequester folliculin, resulting in the activation of the transcription factor TFE3 . These findings establish TMEM55B as a critical link between catabolic processes, lysosomal repair, and transcriptional responses during oxidative stress conditions.

What applications are TMEM55B antibodies validated for?

TMEM55B antibodies have been validated for multiple research applications, with varying effectiveness depending on the specific antibody clone. According to available data, current TMEM55B antibodies are primarily validated for:

These applications have been documented in multiple publications, with Western Blot being the most frequently cited application across research papers . When selecting an antibody for a specific application, researchers should review the validation data provided by manufacturers and consider pilot experiments to determine optimal conditions for their particular experimental system.

What species reactivity should researchers expect from commercial TMEM55B antibodies?

Commercial TMEM55B antibodies demonstrate cross-reactivity with multiple species, though the degree of reactivity may vary between different antibody products. Based on available validation data, researchers can expect:

• Confirmed reactivity with human, mouse, and rat TMEM55B

• Reported reactivity with monkey TMEM55B in published literature

This cross-species reactivity is supported by the high conservation of the TMEM55B protein across mammalian species, particularly in functional domains such as the PPXY motif, which has been documented to be conserved not only in mammals but also in fish and fly models . When working with species not listed in the validated reactivity panel, researchers should perform preliminary validation experiments before proceeding with full-scale studies.

What molecular weight should TMEM55B appear at in Western blot analysis?

When conducting Western blot analysis of TMEM55B, researchers should expect to observe bands at the following molecular weights:

• Calculated molecular weight: 29 kDa (based on the 277 amino acid sequence)

• Observed molecular weight range: 29-32 kDa

The variation between calculated and observed molecular weights may be attributed to post-translational modifications, particularly phosphorylation. Research has demonstrated that TMEM55B undergoes phosphorylation in response to oxidative stress, specifically at residues T111 and S162 . This phosphorylation can be observed as a shift in electrophoretic mobility, resulting in higher molecular weight bands on Western blots . To confirm that higher molecular weight bands represent phosphorylated TMEM55B, researchers can treat lysates with lambda phosphatase prior to electrophoresis, which should eliminate the higher molecular weight bands if they indeed represent phosphorylated forms .

How can researchers optimize immunoprecipitation protocols for studying TMEM55B interactions?

Optimizing immunoprecipitation (IP) protocols for TMEM55B requires careful consideration of both basal and stress-induced protein interactions. Based on published methodologies, the following approach is recommended:

For basal interaction studies:

• Use 0.5-4.0 μg of TMEM55B antibody for every 1.0-3.0 mg of total protein lysate

• Include appropriate controls (IgG control, lysate from TMEM55B knockout cells) to verify specificity

• Consider mild lysis conditions (1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions

For stress-induced interactions:

• Treatment with sodium arsenite (NaAsO₂) has been documented to induce specific interactions between TMEM55B and proteins including PLEKHM1 and VPS41

• The interaction timing is critical - for NaAsO₂-induced interactions, a 2-hour treatment period has been effective in published studies

• For detecting ubiquitination of TMEM55B, denaturing conditions during immunoprecipitation are essential to disrupt non-covalent interactions

For reverse immunoprecipitation validation:

• Confirm interactions by performing reverse IP (e.g., pull down PLEKHM1-Flag and probe for TMEM55B)

• Co-localization studies can support IP findings - TMEM55B co-localizes with interaction partners on Rab7-positive puncta

These methodological refinements are particularly important as TMEM55B interactions show high specificity to certain stress conditions. For example, while NaAsO₂ treatment induces PLEKHM1 binding, other stressors including EBSS, H₂O₂, CCCP, LLOMe, tunicamycin, and thapsigargin do not induce this specific interaction .

What experimental approaches are most effective for studying TMEM55B phosphorylation?

TMEM55B undergoes phosphorylation in response to specific cellular stresses, particularly oxidative stress. To effectively study these phosphorylation events, researchers should consider the following methodological approaches:

• Inducing phosphorylation: Treatment with sodium arsenite (NaAsO₂) has been shown to effectively induce TMEM55B phosphorylation, while other stressors like H₂O₂, CCCP, LLOMe, tunicamycin, and thapsigargin do not trigger the same modification pattern

• Detecting phosphorylation by mobility shift:

  • TMEM55B phosphorylation can be observed as a shift in electrophoretic mobility on Western blots

  • This shift is specifically observed after NaAsO₂ treatment but not with other stress inducers

• Confirming phosphorylation status:

  • Treat immunoprecipitated TMEM55B with Lambda phosphatase

  • The higher molecular weight band should disappear after phosphatase treatment, confirming its identity as phosphorylated TMEM55B

• Identifying phosphorylation sites:

  • Mass spectrometry analysis has identified T111 and S162 as TMEM55B residues that undergo phosphorylation in response to NaAsO₂

  • Site-directed mutagenesis of these residues (T111A, S162A) can be employed to study the functional significance of phosphorylation at these specific sites

• Studying phosphorylation-dependent interactions:

  • TMEM55B phosphorylation appears to regulate its interaction partners

  • For example, phosphorylated TMEM55B interacts with PLEKHM1 and VPS41 while releasing JIP4

  • Co-immunoprecipitation experiments comparing wild-type and phosphorylation-deficient mutants can elucidate the role of phosphorylation in these interaction dynamics

When designing experiments to study TMEM55B phosphorylation, researchers should be mindful that different cellular stressors lead to distinct phosphorylation patterns and downstream interactions, suggesting that TMEM55B may integrate various stress signals through differential post-translational modifications.

How can researchers effectively study TMEM55B's role in autophagy and lysosomal repair?

Investigating TMEM55B's multifaceted role in autophagy and lysosomal repair requires a comprehensive experimental approach that addresses its various functional domains and interaction partners. Based on current research findings, the following methodological framework is recommended:

• Genetic manipulation approaches:

  • Generate TMEM55B knockout cell lines using CRISPR/Cas9

  • Create rescue lines expressing wild-type TMEM55B or mutants (P66A mutation disrupts NEDD4 binding; mutations of the PSAP motif disrupt ESCRT interaction)

  • Develop inducible expression systems to control timing of TMEM55B expression

• Domain-specific functional analysis:

  • The PPXY motif mediates interaction with NEDD4-like E3 ubiquitin ligases

  • Study P66A mutant (proline to alanine mutation in the PPXY motif) to investigate NEDD4-dependent functions

  • Examine the PSAP motif-dependent recruitment of ESCRT machinery for lysosomal repair functions

• Autophagy flux assessment:

  • Monitor LC3-II levels with and without lysosomal inhibitors in TMEM55B-manipulated cells

  • Use tandem mRFP-GFP-LC3 reporters to assess autophagosome-lysosome fusion

  • Measure p62/SQSTM1 degradation rates as an indicator of autophagic degradation

• Lysosomal repair quantification:

  • Induce lysosomal damage using LLOMe or other lysosomotropic agents

  • Assess lysosomal membrane permeabilization using galectin-3 puncta formation

  • Measure calcium-dependent recruitment of ESCRT components to damaged lysosomes

  • Compare repair kinetics between wild-type and TMEM55B-deficient cells

• Stress-specific experimental design:

  • Different stressors engage distinct TMEM55B functions - oxidative stress (NaAsO₂) specifically induces TMEM55B phosphorylation and alters its interaction network

  • Design time-course experiments to capture the dynamics of TMEM55B-dependent responses

  • Compare responses to different types of cellular stress (oxidative, ER, mitochondrial)

• Visualization techniques:

  • Use super-resolution microscopy to visualize TMEM55B localization at lysosomes

  • Perform live-cell imaging with fluorescently tagged TMEM55B to track dynamics during stress responses

  • Implement proximity labeling approaches (BioID, APEX) to identify the TMEM55B proximal proteome under different conditions

By systematically applying these approaches, researchers can dissect the specific contributions of TMEM55B to autophagy regulation, lysosomal repair, and cellular adaptation to stress conditions.

What are the key considerations for studying TMEM55B protein-protein interactions?

TMEM55B engages in a complex network of protein-protein interactions that are dynamically regulated by cellular stress conditions. When investigating these interactions, researchers should consider the following methodological aspects:

• Stress-dependent interaction dynamics:

  • TMEM55B interacts with different partners depending on cellular stress conditions

  • Sodium arsenite (NaAsO₂) specifically induces interactions with PLEKHM1 and VPS41, while causing dissociation from JIP4

  • Other stressors (EBSS, H₂O₂, CCCP, LLOMe, tunicamycin, thapsigargin) do not induce the same interaction patterns

• Domain-specific interactions:

  • The PPXY motif mediates interactions with NEDD4-family E3 ubiquitin ligases

  • Mutation of proline 66 to alanine (P66A) abolishes binding to NEDD4

  • The PSAP motif facilitates interactions with ESCRT complex components

• Validation through multiple approaches:

  • Combine co-immunoprecipitation with reverse immunoprecipitation

  • Support biochemical data with co-localization studies

  • For TMEM55B/PLEKHM1 interactions, co-localization occurs at Rab7-positive structures

• Ubiquitination analysis:

  • Examine TMEM55B ubiquitination under denaturing conditions

  • Seven ubiquitination sites have been identified in the cytosolic domain: K96, K103, K114, K120, K121, K134, and K148

  • Compare ubiquitination patterns between wild-type and P66A mutant TMEM55B

• Phosphorylation-dependent interactions:

  • TMEM55B phosphorylation at T111 and S162 correlates with altered protein interactions

  • Design phosphomimetic (T/S to D/E) and phosphodeficient (T/S to A) mutants to probe the role of phosphorylation in mediating specific interactions

• Experimental setup considerations:

  • Cell type selection: TMEM55B interaction patterns have been documented in U2OS and HeLa cells

  • Lysis conditions: use mild detergents to preserve interactions

  • Timing: interaction dynamics may vary with duration of stress exposure

• Proteomic approaches:

  • Implement proximity labeling methods (BioID, APEX) to identify proximal proteins

  • Perform quantitative proteomics on TMEM55B immunoprecipitates under different conditions

  • Analyze post-translational modifications by mass spectrometry after enrichment steps

By carefully considering these aspects, researchers can effectively map the dynamic interaction network of TMEM55B and understand how these interactions contribute to its roles in cellular stress responses, autophagy regulation, and lysosomal repair.

How can researchers resolve potential antibody cross-reactivity issues in TMEM55B studies?

When working with TMEM55B antibodies, addressing potential cross-reactivity is crucial for ensuring experimental validity. The following methodological approach helps researchers effectively validate antibody specificity and resolve cross-reactivity issues:

• Genetic validation controls:

  • Generate TMEM55B knockout cell lines using CRISPR/Cas9 technology

  • Perform siRNA-mediated knockdown of TMEM55B

  • Include these genetic controls in all key experiments to confirm signal specificity

• Antibody validation matrix:

  • Test multiple TMEM55B antibodies targeting different epitopes

  • Compare commercially available antibodies (e.g., Proteintech 23992-1-AP, Atlas Antibodies HPA048528)

  • Validate each antibody across multiple applications (WB, IP, IHC, ICC-IF)

• Species-specific considerations:

  • While TMEM55B antibodies show reactivity with human, mouse, and rat samples , validation should be performed for each species

  • For evolutionary distant species, perform sequence alignment to assess epitope conservation

  • Consider generating species-specific antibodies if working with non-standard model organisms

• Application-specific validation:

  • For Western blot: confirm band disappearance in knockout/knockdown samples

  • For immunoprecipitation: perform mass spectrometry on immunoprecipitated material

  • For immunofluorescence: include co-localization with known markers (e.g., LAMP1 for lysosomal localization)

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide or recombinant TMEM55B

  • True TMEM55B signal should be significantly reduced or eliminated

  • Non-specific signals will remain unchanged

• Cross-reactivity analysis:

  • Test antibody reactivity against related family members (e.g., TMEM55A)

  • Perform immunoprecipitation followed by mass spectrometry to identify potential cross-reactive proteins

  • Analyze antibody recognition of specific TMEM55B post-translational modifications

• Documentation of antibody validation:

  • Maintain detailed records of validation experiments

  • Document lot-to-lot variation for commercial antibodies

  • Follow reporting standards for antibody validation (e.g., RRID identifiers)

Implementing this systematic validation approach not only ensures experimental reliability but also contributes to the reproducibility of TMEM55B research across different laboratories and experimental systems.

How does TMEM55B respond to different cellular stress conditions?

TMEM55B exhibits differential responses to various cellular stress conditions, with particularly pronounced changes observed during oxidative stress. Understanding these stress-specific responses is essential for designing appropriate experimental paradigms:

• Oxidative stress (NaAsO₂) responses:

  • Induces TMEM55B phosphorylation at residues T111 and S162

  • Causes electrophoretic mobility shift detectable by Western blot

  • Enhances interaction with NEDD4L E3 ubiquitin ligases

  • Promotes TMEM55B ubiquitination at multiple lysine residues (K96, K103, K114, K120, K121, K134, K148)

  • Triggers the formation of TMEM55B interaction with PLEKHM1 and VPS41

  • Disrupts the interaction between TMEM55B and JIP4

  • Induces clustering of TMEM55B-positive lysosomes

• Response specificity:

  • Other cellular stressors (EBSS, H₂O₂, CCCP, LLOMe, tunicamycin, thapsigargin) do not induce the same pattern of TMEM55B modifications and interactions

  • This stress-specific response suggests that TMEM55B may function as a sensor for particular types of cellular damage

• Experimental measurement approaches:

  • Monitor phosphorylation state using phosphatase treatment and mobility shift analysis

  • Track ubiquitination using denaturing immunoprecipitation followed by ubiquitin immunoblotting

  • Assess changes in protein-protein interactions through co-immunoprecipitation experiments

  • Visualize lysosomal clustering and TMEM55B localization through immunofluorescence microscopy

These distinct stress-dependent responses position TMEM55B as a critical regulatory node that integrates specific stress signals to coordinate appropriate cellular responses, particularly in the context of autophagy regulation and lysosomal function. When designing experiments to study TMEM55B function, researchers should carefully consider which stress paradigm best aligns with their specific research questions.

What methodological approaches can distinguish between the multiple functional roles of TMEM55B?

TMEM55B exhibits multiple functional roles in cellular homeostasis, including autophagy regulation, lysosomal repair, and stress signaling. To effectively distinguish between these functions, researchers should implement the following methodological approaches:

• Domain-specific mutant analysis:

  • PPXY motif mutant (P66A): disrupts interaction with NEDD4-family E3 ligases

  • PSAP motif mutant: impairs interaction with ESCRT components

  • Phosphorylation site mutants (T111A, S162A): affects stress-induced protein interactions

  • Compare phenotypes of these mutants to dissect domain-specific functions

• Function-specific assays:

  Function	Assay Approach	Readout
  Autophagy regulation	LC3-II turnover assay with/without Bafilomycin A1	Western blot quantification
  	RFP-GFP-LC3 reporter	Fluorescence microscopy
  	p62/SQSTM1 degradation	Western blot quantification
  Lysosomal repair	Galectin-3 puncta formation	Immunofluorescence
  	Lysosomal pH recovery	LysoSensor imaging
  	ESCRT recruitment kinetics	Live-cell imaging
  TFEB/TFE3 activation	Nuclear translocation	Immunofluorescence
  	Target gene expression	qRT-PCR

• Temporal separation of functions:

  • Design time-course experiments to distinguish early vs. late functions

  • Use inducible expression systems for temporal control of TMEM55B

  • Track sequential molecular events following stress induction

• Biochemical separation of functions:

  • Perform subcellular fractionation to separate lysosomal, cytosolic, and nuclear pools of TMEM55B

  • Isolate TMEM55B-containing protein complexes using size exclusion chromatography

  • Use proximity labeling approaches (BioID, APEX) under different conditions to identify compartment-specific interaction partners

• Genetic interaction studies:

  • Perform genetic epistasis experiments with components of autophagy machinery

  • Test genetic interactions with ESCRT complex components

  • Examine interactions with TFEB/TFE3 signaling pathway components

• Pharmacological dissection:

  • Use kinase inhibitors to block TMEM55B phosphorylation

  • Apply proteasome inhibitors to prevent degradation of ubiquitinated targets

  • Employ lysosomal inhibitors to distinguish between autophagy initiation and flux

By systematically applying these approaches, researchers can disentangle the multiple functions of TMEM55B and determine how these functions are coordinated in response to cellular stress conditions. This comprehensive analysis will contribute to a more nuanced understanding of TMEM55B's role in cellular homeostasis.

What are the optimal conditions for immunofluorescence staining of TMEM55B?

Successfully visualizing TMEM55B through immunofluorescence requires optimization of several technical parameters. Based on published methodologies and antibody validation data, the following protocol recommendations can guide researchers in obtaining specific and robust TMEM55B staining:

• Fixation methods:

  • Paraformaldehyde (4%) fixation for 10-15 minutes at room temperature preserves TMEM55B epitopes while maintaining cellular architecture

  • Avoid methanol fixation as it may disrupt membrane protein epitopes

• Permeabilization options:

  • For visualizing lysosomal TMEM55B: 0.1% Triton X-100 or 0.1% saponin

  • For preserved membrane structures: lower concentrations (0.05%) or alternative detergents like digitonin

• Blocking conditions:

  • 5% normal serum (matched to secondary antibody host) in PBS with 0.1% Triton X-100

  • 1-3% BSA can be used as an alternative blocking agent

  • Include 0.1% Tween-20 to reduce background

• Antibody selection and dilution:

  • Starting dilution range: 1:100 to 1:500 for primary antibodies

  • Optimize through titration experiments for each specific application

  • Consider testing multiple antibodies targeting different TMEM55B epitopes

• Co-localization markers:

  • LAMP1 or LAMP2 for lysosomal co-localization

  • Rab7 for late endosomal/lysosomal localization

  • In stress conditions, co-stain for interaction partners like PLEKHM1

• Signal enhancement strategies:

  • Tyramide signal amplification for weak signals

  • Consider using directly conjugated primary antibodies to reduce background

  • Extended primary antibody incubation (overnight at 4°C) may improve signal-to-noise ratio

• Imaging considerations:

  • Confocal microscopy recommended for precise lysosomal localization

  • Super-resolution techniques (STED, SIM, STORM) for detailed subcellular distribution

  • Live-cell imaging with fluorescently tagged TMEM55B for dynamic studies

• Validation controls:

  • TMEM55B knockout or knockdown cells as negative controls

  • Pre-absorption of antibody with immunizing peptide to verify specificity

  • Secondary-only controls to assess non-specific binding

By systematically optimizing these parameters, researchers can achieve reliable and specific immunofluorescence staining of TMEM55B for various experimental applications, including localization studies, stress response analyses, and co-localization with interaction partners.

How can researchers effectively use TMEM55B antibodies for studies across different species?

Working with TMEM55B antibodies across multiple species requires careful consideration of epitope conservation and validation strategies. The following methodological approach facilitates effective cross-species application:

• Epitope conservation analysis:

  • TMEM55B shows high conservation across mammalian species

  • The PPXY motif is particularly conserved, present in mammals, fish, and flies

  • Before applying antibodies to non-validated species, perform sequence alignment to assess epitope conservation

• Validated species reactivity:

  • Commercial antibodies have confirmed reactivity with human, mouse, and rat TMEM55B

  • Reported reactivity with monkey TMEM55B in published literature

• Cross-species validation strategy:

  • Western blot validation: confirm band at expected molecular weight (29-32 kDa)

  • Genetic validation: test antibody in TMEM55B knockout tissues/cells from each species

  • Immunoprecipitation-mass spectrometry: confirm antibody pulls down TMEM55B from multiple species

• Application-specific considerations:

  Application	Cross-species Adaptation
  Western Blot	Adjust sample loading (20-50 μg total protein)
  	Optimize primary antibody dilution (1:2000-1:10000)
  	Consider species-specific detection systems
  Immunohistochemistry	Optimize antigen retrieval for each species
  	Test multiple fixation protocols
  	Validate with species-specific positive controls
  Immunoprecipitation	Adjust antibody amount (0.5-4.0 μg) based on target abundance
  	Optimize lysis conditions for each tissue/cell type

• Species-specific background mitigation:

  • Pre-absorb antibodies with acetone powder from the relevant species

  • Use species-matched normal IgG for negative controls

  • Consider direct conjugation to minimize secondary antibody cross-reactivity

• Evolutionary divergence considerations:

  • For distantly related species, consider generating species-specific antibodies

  • When using commercial antibodies, contact manufacturers for unpublished cross-reactivity data

  • Perform parallel validation with multiple antibodies targeting different TMEM55B epitopes

By implementing this systematic approach to cross-species application, researchers can confidently extend TMEM55B studies across multiple model organisms while maintaining experimental rigor and reproducibility.

What are the emerging research directions in TMEM55B biology?

TMEM55B research is evolving rapidly, with several promising directions emerging from recent discoveries about its multifunctional roles in cellular homeostasis. Based on current literature, the following represent key areas for future investigation:

• Integration of stress responses:

  • TMEM55B appears to respond specifically to certain stressors (particularly NaAsO₂) but not others

  • Future research should elucidate how TMEM55B distinguishes between different stress types

  • The kinase(s) responsible for stress-induced TMEM55B phosphorylation remain to be identified

• Therapeutic implications in lysosomal disorders:

  • Given TMEM55B's role in lysosomal repair and autophagy regulation , it represents a potential therapeutic target for lysosomal storage disorders

  • Modulation of TMEM55B activity might enhance cellular clearance mechanisms

  • Development of small molecules targeting TMEM55B functional domains could provide new therapeutic avenues

• Tissue-specific functions:

  • TMEM55B expression and function across different tissues remains incompletely characterized

  • Investigation of tissue-specific interactomes may reveal specialized roles

  • Potential for tissue-specific regulation of autophagy and lysosomal function

• Relationship with other membrane trafficking regulators:

  • TMEM55B interacts with multiple trafficking components including PLEKHM1, VPS41, and JIP4

  • The hierarchical organization of these interactions and their regulatory mechanisms merit further investigation

  • The mutual exclusivity of certain interactions (e.g., PLEKHM1 vs. JIP4) suggests complex regulatory mechanisms

• Role in transcriptional regulation:

  • TMEM55B influences TFE3 activity through folliculin sequestration

  • The full extent of TMEM55B's impact on transcriptional networks remains to be explored

  • Potential crosstalk with other transcriptional programs beyond TFEB/TFE3 pathways

Future research addressing these emerging areas will likely require interdisciplinary approaches combining structural biology, advanced imaging, systems biology, and in vivo models. These efforts will continue to uncover the complex roles of TMEM55B in cellular homeostasis and may ultimately lead to novel therapeutic strategies for diseases involving lysosomal dysfunction and impaired autophagy.

What methodological advances are needed to address current limitations in TMEM55B research?

Despite significant progress in understanding TMEM55B biology, several methodological limitations constrain further advances. Addressing these challenges requires innovative approaches:

• Structural characterization limitations:

  • Current understanding of TMEM55B structure remains limited

  • Development of strategies for membrane protein crystallization or cryo-EM approaches for TMEM55B structural determination

  • Implementation of computational modeling informed by evolutionary constraints and cross-linking mass spectrometry

• Dynamic interaction monitoring:

  • Current interaction studies provide static snapshots rather than dynamic information

  • Implementation of FRET/BRET biosensors to monitor TMEM55B interactions in real-time

  • Development of optogenetic tools to temporally control TMEM55B function and localization

  • Application of live-cell proximity labeling approaches with rapid kinetics

• Tissue-specific function assessment:

  • Current understanding derives primarily from cell culture models

  • Generation of conditional TMEM55B knockout mouse models for tissue-specific studies

  • Development of tissue-clearing techniques compatible with TMEM55B immunostaining

  • Implementation of spatial transcriptomics to correlate TMEM55B expression with tissue-specific functions

• Post-translational modification mapping:

  • Comprehensive characterization of TMEM55B modifications beyond phosphorylation and ubiquitination

  • Development of modification-specific antibodies for stress-induced TMEM55B phosphorylation

  • Application of targeted proteomics approaches for quantitative PTM profiling across conditions

• Functional domain dissection:

  • Refinement of domain-specific mutants beyond current PPXY and phosphorylation site mutations

  • Development of domain-specific intrabodies for acute inhibition

  • Implementation of protein complementation approaches to study domain-specific interactions