TRIM28 Monoclonal Antibody

What is TRIM28 and why is it significant in research?

TRIM28 (Tripartite Motif Containing 28), also known as KAP1 (KRAB-associated protein 1) or TIF1β (Transcription Intermediary Factor 1-beta), is a ubiquitously expressed protein involved in numerous critical cellular functions. It serves as a molecular scaffold that recruits chromatin-modifying complexes to specific genomic loci.

TRIM28 is significant in research due to its involvement in:

• Transcriptional regulation and epigenetic silencing

• Cellular differentiation and proliferation

• DNA damage repair pathways

• Viral suppression mechanisms

• Apoptosis regulation

• Cancer development and metastasis

• Immune response modulation

The protein contains multiple functional domains: RING, B-box, coiled-coil (collectively known as RBCC), and a C-terminal region with PHD and bromodomain motifs, enabling its diverse cellular activities .

What are the primary cellular functions of TRIM28?

TRIM28 functions primarily as:

• Transcriptional corepressor: Recruits histone deacetylases and histone methyltransferases to gene promoters through interactions with KRAB-domain zinc finger proteins, leading to heterochromatin formation and gene silencing .

• SUMO E3 ligase: Mediates SUMOylation of various proteins, including CDK9, affecting their function and stability .

• Epigenetic regulator: Controls DNA methylation patterns and contributes to silencing of endogenous retroviruses and retrotransposons .

• Modulator of DNA damage response: Participates in DNA repair mechanisms through protein-protein interactions and post-translational modifications .

• Regulator of stem cell properties: Essential for embryonic development and maintenance of stem cell pluripotency, as evidenced by embryonic lethality in TRIM28 knockout mice .

Recent research has revealed that TRIM28 promotes chemokine-driven recruitment of myeloid-derived suppressor cells (MDSCs) through RIPK1-mediated NF-κB activation in cancer microenvironments .

How does the domain structure of TRIM28 relate to its function?

TRIM28's multidomain structure directly dictates its functional versatility:

Different domains serve specific roles in TRIM28 functions. For instance:

• The B-box domain has been shown to increase L1 retrotransposition and facilitate shorter cDNA generation .

• The SUMO E3 ligase activity resides primarily in the RING domain, which is essential for SUMOylating targets like CDK9 to regulate HIV-1 latency .

• The coiled-coil domain mediates protein-protein interactions necessary for TRIM28's scaffolding functions .

Mutations in specific domains produce distinct functional outcomes. For example, the B-box A160D, T163A, and E175R mutations impact TRIM28's ability to regulate L1 retrotransposition .

What criteria should be considered when selecting a TRIM28 monoclonal antibody?

When selecting a TRIM28 monoclonal antibody for research applications, consider:

• Epitope specificity: Choose antibodies that target conserved regions of TRIM28 or specific domains based on your research question. Antibodies recognizing the N-terminal RBCC region are often used for general detection, while domain-specific antibodies may be needed for specialized applications.

• Validated applications: Ensure the antibody is validated for your specific application (Western blot, immunoprecipitation, ChIP, immunohistochemistry, flow cytometry, etc.) .

• Species reactivity: Verify cross-reactivity with your experimental model. Many TRIM28 antibodies react with human, mouse, and rat TRIM28 due to high sequence conservation .

• Clone information: Monoclonal antibodies from different clones (e.g., 9E3, 3H2) may have different properties and application suitability .

• Post-translational modification detection: For studying phosphorylated forms of TRIM28, specific phospho-antibodies (e.g., against phospho-S824 or phospho-S473) may be required .

• Publication history: Prioritize antibodies with established track records in peer-reviewed literature that match your experimental conditions.

How should TRIM28 antibodies be validated for research applications?

Thorough validation of TRIM28 antibodies should include:

• Positive and negative controls:

  • Positive control: Cell lines known to express high levels of TRIM28 (HeLa, PC-3, HEK293, A549, Jurkat, THP-1)

  • Negative control: TRIM28 knockout cell lines (e.g., U2OS KO cell line)

  • Knockdown validation: Comparing antibody signal in TRIM28-depleted vs. wild-type cells

• Multiple application validation:

  • Western blot: Verify a single specific band at approximately 100 kDa

  • Immunoprecipitation: Confirm ability to pull down TRIM28 and associated proteins

  • Immunohistochemistry/Immunofluorescence: Evaluate predominantly nuclear staining pattern

  • ChIP: Validate enrichment at known TRIM28 binding sites (e.g., retroviral LTRs)

• Cross-reactivity assessment: Test for non-specific binding to other TRIM family proteins, particularly other TIF1 family members.

• Reproducibility evaluation: Ensure consistent results across different experimental conditions and cell/tissue types.

Example from literature: Antibody validation was demonstrated in Figure 7 of one study, where flow cytometry analysis of A549 cells stained with anti-TRIM28 antibody showed clear positive staining compared to isotype control and unlabeled samples .

What controls should be included when using TRIM28 antibodies in experiments?

For robust experimental design with TRIM28 antibodies, include these controls:

• Isotype control: Include appropriate isotype-matched control antibody to assess non-specific binding .

• Genetic controls:

  • TRIM28 knockout or knockdown cells/tissues (e.g., using shRNA or CRISPR-Cas9)

  • Cells with re-expressed TRIM28 in knockout background for rescue experiments

  • Domain mutants to validate domain-specific functions

• Loading controls: For Western blots, include housekeeping proteins (GAPDH, β-actin) to normalize protein loading .

• Subcellular fractionation controls: When examining TRIM28 in specific cellular compartments, include markers for nuclear (e.g., histone H3) and cytoplasmic (e.g., GAPDH) fractions.

• ChIP controls:

  • Input control (non-immunoprecipitated chromatin)

  • IgG control for non-specific binding

  • Positive control loci (known TRIM28 binding sites like retroviral LTRs)

• Peptide competition: Pre-incubation of antibody with the immunizing peptide should abolish specific signal.

Example approach: "ChIP assay was performed with anti-TRIM28 and the control IgG antibodies... HT1080 cells were infected with PFV and collected for ChIP assay with anti-TRIM28 antibody 48 h later. The purified DNA eluate was quantified by qPCR."

How can TRIM28 antibodies be optimized for Western blot analysis?

For optimal Western blot detection of TRIM28:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors

  • For detecting SUMO-modified forms, include N-ethylmaleimide (20 mM) to inhibit SUMO proteases

  • Heat samples at 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol

• Gel electrophoresis conditions:

  • Use 8-10% SDS-PAGE gels to achieve good resolution of TRIM28 (~100 kDa)

  • Load 20-50 μg of total protein per lane

  • Include molecular weight markers spanning 75-150 kDa range

• Transfer parameters:

  • Transfer proteins to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Verify transfer efficiency with reversible protein staining

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature

  • Incubate with anti-TRIM28 primary antibody at 0.5-1 μg/mL overnight at 4°C

  • Wash with TBS-0.1% Tween three times, 5 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:10,000 dilution) for 1.5 hours at room temperature

  • Develop using enhanced chemiluminescence (ECL) detection

Practical tip: "After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-KAP1/TRIM28 antigen affinity purified monoclonal antibody at 0.5 μg/mL overnight at 4°C..."

What are the best practices for immunoprecipitation using TRIM28 antibodies?

For successful immunoprecipitation (IP) of TRIM28 and its interacting partners:

• Lysis buffer selection:

  • Use NP-40 or CHAPS-based lysis buffers for preserving protein-protein interactions

  • Include protease inhibitors, phosphatase inhibitors, and NEM (for SUMO-modified proteins)

  • Example buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitor cocktail

• Pre-clearing:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Reserve 5-10% of lysate as input control

• Antibody binding:

  • Use 2-5 μg of TRIM28 antibody per 500 μg of total protein

  • Incubate overnight at 4°C with gentle rotation

  • For tagged TRIM28, anti-tag antibodies (e.g., anti-FLAG) can be used

• Bead capture and washing:

  • Add protein A/G beads and incubate for 1-3 hours at 4°C

  • Wash beads 3-5 times with washing buffer (lysis buffer with reduced detergent)

  • For co-IP of specific partners, adjust salt and detergent concentrations to maintain interactions

• Elution and analysis:

  • Elute proteins by boiling in Laemmli buffer

  • Analyze by Western blot, probing for TRIM28 and potential interacting partners

Example protocol: "To test whether human and mouse TRIM28 proteins interact with human L1 ORF1 or ORF2 protein, we transiently co-expressed FLAG-tagged H-TRIM28 or M-TRIM28 with L1 ORF1p or T7-tagged L1 ORF2p and performed co-immunoprecipitation in HeLa cells using beads conjugated with anti-FLAG antibodies."

How can TRIM28 localization be studied using immunofluorescence techniques?

For optimal immunofluorescence (IF) detection of TRIM28:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • For nuclear protein access, permeabilize with 0.2% Triton X-100 for 10 minutes

• Antigen retrieval options:

  • For formaldehyde-fixed samples, perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

  • For tissue sections, enzymatic antigen retrieval may be used

  • For some applications, methanol fixation (-20°C, 10 min) may provide better nuclear antigen accessibility

• Blocking and antibody incubation:

  • Block with 10% goat serum for 1 hour at room temperature

  • Incubate with TRIM28 primary antibody (1-2 μg/mL) overnight at 4°C

  • Wash 3× with PBS

  • Incubate with fluorophore-conjugated secondary antibody (e.g., DyLight 488) at 1:100-1:500 dilution for 30-60 minutes

  • Counterstain nucleus with DAPI

  • Mount with anti-fade mounting medium

• Microscopy settings:

  • Use appropriate filter sets for the fluorophores used

  • TRIM28 typically shows predominantly nuclear localization with some nucleolar exclusion

  • Capture Z-stacks for precise localization analysis

Example approach: "TRIM28 was detected in immunocytochemical section of U20S cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL mouse anti-TRIM28 Antibody overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C."

How does TRIM28 contribute to cancer progression and metastasis?

TRIM28 plays multiple roles in cancer development and progression:

• Immunotherapy resistance mechanisms:

  • TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer

  • Functions through RIPK1-mediated NF-κB activation

  • Drives recruitment of immunosuppressive myeloid-derived suppressor cells (MDSCs)

  • Silencing TRIM28 enhances efficacy of anti-PD-1 immunotherapy by reshaping the tumor microenvironment

These findings suggest that targeting TRIM28 may represent a promising therapeutic strategy for cancer treatment, particularly in combination with immunotherapy approaches.

What is the role of TRIM28 in viral restriction and latency?

TRIM28 plays crucial but complex roles in viral restriction and latency, acting through multiple mechanisms:

• Retroviral silencing:

  • TRIM28 mediates silencing of murine leukemia viruses (MLVs) and related retroelements in embryonic cells

  • Targets both Pro tRNA primer binding site (PBS Pro) and Lys-1,2 tRNA primer binding site (PBS Lys-1,2)

  • Functions as a transcriptional corepressor by recruiting the NuRD histone deacetylase complex, histone H3 K9 methyltransferase ESET, and heterochromatin-associated protein HP1

  • DNA-binding proteins recruit TRIM28 to viral sequences, as demonstrated by EMSA and ChIP assays

• HIV-1 latency regulation:

  • TRIM28 suppresses HIV-1 expression through both SUMO E3 ligase activity and epigenetic adaptor function

  • SUMOylates CDK9 (P-TEFb catalytic subunit) at Lys44, Lys56, and Lys68 with SUMO4

  • This SUMOylation inhibits CDK9 kinase activity or prevents P-TEFb assembly by blocking interaction between CDK9 and Cyclin T1

  • Enriched on HIV-1 LTR compared to host-provirus junction and viral coding regions

  • Depletion of TRIM28 decreases H3K9me2/3 and increases H3K4me3/H3K9Ac on HIV-1 LTR

• Prototype foamy virus (PFV) restriction:

  • TRIM28 acts as a restriction factor for PFV replication

  • Significantly enriched in the R, U5, and PBS regions of the PFV LTR promoter

  • Interacts with viral protein Tas to inhibit its transactivation function

  • Overexpression of TRIM28 suppresses PFV viral load and transcription

• LINE-1 retrotransposition:

  • Unlike its typical repressive function, TRIM28 increases L1 retrotransposition through its B-box domain

  • Facilitates shorter cDNA and L1 insert generation in cultured cells

  • Interacts with L1 ORF2 protein but not ORF1 protein

  • This function appears distinct from TRIM28's usual gene silencing activities

These diverse roles highlight TRIM28's multifunctional nature in viral restriction and activation, making it a potential target for developing treatments for viral latency, particularly in HIV-1 infection.

How does post-translational modification of TRIM28 regulate its functions?

TRIM28 undergoes multiple post-translational modifications that critically regulate its diverse cellular functions:

• Phosphorylation:

  • Ser473 phosphorylation: Mediated by EIF2AK2/PKR during inflammatory responses, promoting TRIM28-CTIF interaction and inhibiting aggresome formation

  • Ser824 phosphorylation: Occurs in response to DNA damage, altering TRIM28's interaction with chromatin and its transcriptional repression activities

  • More aggressive breast cancer subtypes show higher levels of TRIM28-S824-phospho compared to luminal A subtype

• SUMOylation:

  • TRIM28 undergoes intramolecular SUMOylation of its bromodomain

  • This modification is crucial for recruiting chromatin-modifying enzymes and transcriptional repression

  • Functions as a SUMO E3 ligase for other proteins (e.g., CDK9, IRF7)

  • SUMO modification sites include particular lysine residues in the bromodomain region

• Ubiquitination:

  • TRIM28 possesses E3 ubiquitin ligase activity through its RING domain

  • Can mediate K48-linked polyubiquitination leading to protein degradation

  • Also mediates K63-linked polyubiquitination of RIPK1, sustaining NF-κB pathway activation

• Glycosylation:

  • TRIM28 interacts with O-linked β-N-acetylglucosamine transferase (OGT)

  • This interaction occurs specifically in cells with normal genomic methylation patterns

  • OGT transfers N-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls of various chromatin factors

  • This glycosylation both antagonizes protein phosphorylation and induces structural transitions in chromatin factors

The interplay between these modifications creates a complex regulatory network. For example, in HIV-1 latency, TRIM28's SUMOylation activity toward CDK9 contributes to viral suppression, while in inflammatory responses, phosphorylation at Ser473 redirects TRIM28 function toward aggresome regulation. Understanding these modifications is crucial for developing targeted therapeutic approaches in various disease contexts.

What are common challenges when working with TRIM28 antibodies and how can they be overcome?

Researchers frequently encounter these challenges when using TRIM28 antibodies:

• High background in Western blots:

  • Cause: Non-specific binding or excessive antibody concentration

  • Solution: Increase blocking time (2-3 hours), use 5% BSA instead of milk for blocking, optimize primary antibody dilution (start with 1:1000-1:2000), increase wash steps (5× 5 minutes), or try a different blocking buffer

• Weak or absent signal in immunoprecipitation:

  • Cause: Epitope masking due to protein-protein interactions

  • Solution: Try alternative antibody clones targeting different epitopes, modify lysis conditions (adjust salt and detergent concentrations), or perform cross-linking before lysis to stabilize complexes

• Inconsistent ChIP results:

  • Cause: TRIM28 binding can be context-dependent and affected by cell state

  • Solution: Carefully control cell culture conditions, synchronize cells when possible, optimize crosslinking time (8-10 minutes typically works well), ensure sufficient sonication, and include spike-in controls for normalization

• Post-translational modification detection issues:

  • Cause: Modifications like phosphorylation or SUMOylation may be labile

  • Solution: Include phosphatase inhibitors and SUMO protease inhibitors (NEM) in lysis buffers, use phospho-specific antibodies for particular modifications, perform IP under denaturing conditions to preserve modifications

• Nuclear protein extraction difficulties:

  • Cause: TRIM28 is predominantly nuclear and tightly chromatin-bound

  • Solution: Use specialized nuclear extraction buffers with higher salt concentrations (300-400 mM NaCl), include benzonase nuclease treatment to release chromatin-bound proteins

Example troubleshooting approach from literature: "For studying phosphorylated forms of TRIM28, specific phospho-antibodies (e.g., against phospho-S824 or phospho-S473) may be required" and include phosphatase inhibitors in all buffers .

How can specificity of TRIM28 antibody binding be verified in different experimental contexts?

Verifying TRIM28 antibody specificity is critical across experimental platforms:

• Western blot validation:

  • Compare signal between wild-type and TRIM28-knockdown/knockout samples

  • Verify correct molecular weight (approximately 100 kDa)

  • Perform peptide competition assay by pre-incubating antibody with immunizing peptide

  • Test across multiple cell lines with known TRIM28 expression (HeLa, PC-3, HEK293, A549, Jurkat, THP-1)

• Immunoprecipitation validation:

  • Perform reverse IP with antibodies against known TRIM28 interacting partners

  • Confirm co-IP of established TRIM28 binding proteins (HP1, SETDB1, NuRD components)

  • Compare IP efficiency using different antibody clones

  • Perform mass spectrometry on immunoprecipitated material to confirm TRIM28 presence

• ChIP-seq validation:

  • Compare enrichment profiles with published TRIM28 binding patterns

  • Verify enrichment at known TRIM28 targets (endogenous retroviruses, zinc finger protein binding sites)

  • Perform ChIP-qPCR at specific loci before proceeding to sequencing

  • Include IgG controls and analyze non-specific binding

• Immunofluorescence validation:

  • Compare staining pattern between wild-type and TRIM28-depleted cells

  • Verify predominantly nuclear localization with potential nucleolar exclusion

  • Perform co-localization with other nuclear markers

  • Test specificity across different fixation and permeabilization methods

Example validation approach: "To demonstrate that specific depletion of TRIM28 from PCC4 cells by RNAi was responsible for the disappearance of the PBS Lys-1,2 RBS shift, an adenovirus was used to transiently re-express an RNAi-resistant TRIM28-myc/HIS construct in PCC4 111 cells. The transient expression of TRIM28-myc/HIS restored the RBS shift with the 28-bp PBS Lys-1,2 probe..."

What technical considerations are important for studying TRIM28 interactions with binding partners?

When investigating TRIM28's interactions with binding partners, consider these technical aspects:

• Lysis and buffer conditions:

  • Use mild lysis conditions to preserve protein-protein interactions (e.g., 0.5% NP-40 or 0.3% CHAPS)

  • Adjust salt concentration based on expected interaction strength (150-300 mM NaCl)

  • For studying SUMO or ubiquitin modifications, include deubiquitinase inhibitors (PR-619) and SUMO protease inhibitors (NEM, 20 mM)

  • Consider specialized buffers for particular partners (e.g., chromatin-associated proteins may require nuclease treatment)

• Expression system selection:

  • For transient overexpression, consider epitope tags that don't interfere with protein function (small tags like FLAG or HA at C-terminus generally work well)

  • For endogenous interactions, use high-affinity antibodies against TRIM28 or its partners

  • Control expression levels to avoid non-physiological interactions from massive overexpression

• Detection methods optimization:

  • For weak or transient interactions, consider crosslinking approaches (formaldehyde, DSS, or photo-crosslinking)

  • For spatial information, use proximity ligation assay (PLA) to visualize interactions in situ

  • For comprehensive interaction mapping, combine IP with mass spectrometry

  • For direct interaction verification, use purified recombinant proteins in GST pull-down assays

• Domain mapping considerations:

  • Use domain deletion or point mutation constructs to map interaction interfaces

  • Consider the multidomain structure of TRIM28 (RING, B-box, coiled-coil, PHD, bromodomain)

  • Verify that mutations don't disrupt protein folding using circular dichroism or thermal shift assays

Example from the literature: "Immunoprecipitation and GST pull-down assays demonstrated that TRIM28 interacts with TWIST1 directly and this interaction is presumed to protect TWIST1 from degradation" and "Through global site-specific SUMO-MS study and serial SUMOylation assays, we identified that P-TEFb catalytic subunit CDK9 is significantly SUMOylated by TRIM28 with SUMO4" .

How should TRIM28 chromatin immunoprecipitation (ChIP) data be analyzed and interpreted?

Analysis and interpretation of TRIM28 ChIP data requires specific considerations:

• Peak calling and normalization:

  • Use appropriate peak calling algorithms (MACS2, HOMER) with input control normalization

  • For TRIM28 ChIP-seq, use broad peak settings as TRIM28 often binds extended genomic regions

  • Normalize to spike-in controls if available to account for global differences

  • For comparing conditions, use differential binding analysis tools (DiffBind, HOMER getDifferentialPeaks)

• Genomic feature association:

  • Analyze TRIM28 binding relative to genomic features (promoters, enhancers, gene bodies)

  • Examine enrichment at repetitive elements, particularly endogenous retroviruses and LINEs

  • TRIM28 commonly binds retroviral LTRs and tRNA primer binding sites

  • Evaluate co-localization with KRAB-zinc finger protein binding sites

• Integration with epigenetic marks:

  • Correlate TRIM28 binding with repressive histone marks (H3K9me3, H3K27me3)

  • Compare with active chromatin marks (H3K4me3, H3K27ac) to identify differential regulation

  • Analyze DNA methylation patterns at TRIM28 binding sites

  • TRIM28 binding typically correlates with increased H3K9me2/3 and decreased H3K4me3/H3K9Ac

• Motif analysis and co-factor binding:

  • Perform de novo motif discovery on TRIM28 binding sites

  • Look for enrichment of known motifs for KRAB-zinc finger proteins

  • Integrate with ChIP-seq data for known TRIM28 partners (HP1, SETDB1)

  • For viral integration sites, analyze binding relative to viral sequence elements

Example interpretation from literature: "We found that endogenous Trim28 was enriched in the LTR regions, especially significantly enriched in R (tenfold change, p < 0.01), U5 and downstream in the PBS regions (threefold change, p < 0.01)" .

What controls and analytical approaches are needed for interpreting TRIM28 functional studies?

For robust interpretation of TRIM28 functional studies:

• Genetic manipulation controls:

  • Use multiple independent knockdown/knockout approaches (siRNA, shRNA, CRISPR-Cas9)

  • Include rescue experiments with wild-type TRIM28 to confirm specificity

  • Use domain mutants to dissect specific functions (e.g., RING domain mutants for E3 ligase activity)

  • Test multiple cell lines to ensure observed effects aren't cell-type specific

• Transcriptomic analysis approaches:

  • Compare RNA-seq profiles between TRIM28 wild-type, knockdown, and domain mutants

  • Use algorithms like Kallisto and Sleuth for transcript quantification and differential expression

  • Perform gene set enrichment analysis (GSEA) to identify affected pathways

  • Specifically examine expression of repetitive elements and endogenous retroviruses

• Phenotypic assay considerations:

  • For cancer studies, assess multiple aspects of cancer biology (proliferation, migration, invasion, stemness)

  • Use both in vitro and in vivo models for comprehensive understanding

  • For tumor formation studies, perform limiting dilution assays to quantify cancer stem cell frequency

  • Include time-course experiments to distinguish immediate vs. delayed effects

• Statistical analysis requirements:

  • Use appropriate statistical tests with multiple testing correction

  • Report effect sizes and confidence intervals, not just p-values

  • Consider biological replicates (n≥3) across independent experiments

  • For animal studies, ensure proper randomization and blinding

Example analytical approach: "We used RNA-Seq by Expectation-Maximization (RSEM) analysis and gene set enrichment analysis (GSEA) to detect cellular pathways altered in the TRIM28 WT samples compared to the Control and TRIM28 3M samples (N = 4 each). This approach also identified DNA repair as a significantly downregulated pathway when TRIM28 WT was overexpressed."

How can TRIM28 post-translational modifications be accurately detected and quantified?

For precise detection and quantification of TRIM28 post-translational modifications:

• Phosphorylation analysis:

  • Use phospho-specific antibodies for known sites (Ser473, Ser824)

  • Perform phosphatase treatment controls to confirm specificity

  • For large-scale analysis, use phospho-enrichment (TiO2, IMAC) followed by mass spectrometry

  • Quantify using stable isotope labeling (SILAC) or label-free quantification methods

  • Validate key sites with site-specific mutants (S→A or S→D/E mutations)

• SUMOylation detection:

  • Use denaturing conditions during lysis and IP to preserve SUMO modifications

  • Include SUMO protease inhibitors (20 mM NEM)

  • For tagged SUMO, perform Ni-NTA pulldown under denaturing conditions

  • Confirm specific sites using mass spectrometry and lysine-to-arginine mutations

  • Use SUMO-site prediction tools to guide mutation studies

• Ubiquitination analysis:

  • Distinguish between K48-linked (degradative) and K63-linked (signaling) polyubiquitination

  • Use linkage-specific antibodies or UbiCREST assay for linkage determination

  • For enhanced detection, express His-tagged ubiquitin and perform Ni-NTA pulldown

  • Analyze ubiquitination in the presence of proteasome inhibitors (MG132)

• Glycosylation detection:

  • Detect O-GlcNAcylation using specific antibodies or lectins (wheat germ agglutinin)

  • Use enzymatic labeling approaches (GalT labeling with UDP-GalNAz)

  • Confirm specificity with O-GlcNAcase inhibitors (Thiamet G)

  • For site identification, use electron transfer dissociation (ETD) mass spectrometry