SSU72 Antibody

What is SSU72 and what are its primary cellular functions?

SSU72 is a conserved protein phosphatase that catalyzes the dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II. It plays critical roles in multiple cellular processes:

• RNA processing and transcription termination

• T cell receptor (TCR) signaling regulation via ZAP-70 binding

• Fine-tuning of immune responses and T cell differentiation

• Telomere replication termination

• Thermogenic adaptation in brown adipose tissue

• Liver function and protection against hepatocellular carcinoma

The protein has a molecular weight of approximately 22.6-30 kDa and functions in multiple cellular compartments, with activity regulated by various signaling pathways.

How is SSU72 expressed in different tissues and what is its subcellular localization?

SSU72 shows variable expression patterns across tissues and dynamic subcellular localization:

Tissue expression:

• Expressed in multiple mammalian tumor cell lines including C33A, COS7, HeLa, J-45, 293, and MCF7

• Northern blot analysis reveals a doublet transcript pattern with differential expression:

  • Lower transcript predominant in J-45 lymphocytes

  • Upper transcript more abundant in kidney-derived cells (293 and COS7)

Subcellular localization:

• Primary localization in the nucleus and cytoplasm

• Immunohistochemical analysis shows clear plasma membrane localization in addition to nuclear and cytoplasmic expression

• Upon TCR stimulation, SSU72 can translocate to the cell membrane

• In T cells activated with anti-CD3/28, IL-2 and TGFβ, SSU72 frequently exhibits recruitment to the cytoplasmic face of the plasma membrane

This dynamic localization reflects SSU72's diverse functions in transcriptional regulation, signaling, and cell differentiation.

What are the optimal applications and dilutions for SSU72 antibodies?

SSU72 antibodies can be effectively used in several research applications with specific recommended protocols:

When using SSU72 antibodies, include appropriate controls:

• Positive control: MCF-7 or COS-7 cells are suggested positive controls

• Negative control: SSU72 knockdown samples using siRNA or shRNA

• Competition assay: Pre-incubation with recombinant SSU72 protein to verify specificity

Note that some antibodies demonstrate good reactivity with human samples but poor performance with mouse tissues .

Why might I observe different molecular weight bands when using SSU72 antibodies?

Variation in detected SSU72 bands can occur for several reasons:

• Alternative isoforms: SSU72 has at least two isoforms from alternative splicing , which may appear as distinct bands.

• Post-translational modifications: As a phosphatase, SSU72 itself might undergo modification, causing mobility shifts.

• Species variation: Human SSU72 has a reported length of 194 amino acids and theoretical mass of 22.6 kDa, but typically migrates at 27-30 kDa on SDS-PAGE .

• Technical factors:

  • Observed band size varies slightly between studies (23-30 kDa)

  • Some antibodies detect a single band (~30 kDa) in human cell lines

  • Some detection systems may reveal non-specific bands

For proper interpretation, researchers should:

• Verify band identity using SSU72 knockdown controls

• Include recombinant SSU72 protein as a positive control

• Consider using antibodies targeting different epitopes to confirm specificity

How does SSU72 regulate T cell receptor signaling and T cell differentiation?

SSU72 serves as a crucial regulator of T cell receptor signaling and differentiation through multiple mechanisms:

• Direct binding to ZAP-70:

  • SSU72 phosphatase directly binds to ZAP-70, a key initiator of TCR signaling

  • It regulates ZAP-70 tyrosine phosphorylation, providing fine-tuning of TCR signaling

  • Upon TCR stimulation, SSU72-deficient T cells show increased phosphorylation of ZAP-70 and downstream molecules

• T cell differentiation regulation:

  • SSU72 deletion disrupts CD4+ T cell differentiation into regulatory T cells (Tregs)

  • SSU72-deficient T cells produce higher levels of effector cytokines (IL-2, IFNγ)

  • Enhanced production of IFN-γ and IL-4 under TH1 and TH2-polarizing conditions, respectively

  • Markedly fewer iTregs (characterized by Foxp3 expression) develop from SSU72-deficient CD4+ T cells

• PLCγ1 interaction:

  • SSU72 forms a complex with PLCγ1, an essential molecule for TCR signaling and Treg development

  • SSU72 deficiency impairs PLCγ1 downstream signaling, resulting in failure of Foxp3 induction

• T cell homeostasis effects:

  • Deletion of SSU72 in T cells leads to:

    • Defects in thymic development of invariant natural killer T cells

    • Reduced CD4+ and CD8+ T cell numbers in the periphery

    • Increased CD44hiCD62Lomemory T cells and fewer CD44loCD62Lhinaïve T cells

    • Spontaneous inflammation at 6 months of age

These findings establish SSU72 as a critical factor in maintaining appropriate TCR signaling and balanced T cell differentiation.

What is the role of SSU72 in autoimmune diseases and how can it be investigated?

SSU72 plays a significant role in autoimmune disease pathogenesis and shows therapeutic potential:

Autoimmune regulation mechanisms:

• SSU72 attenuates autoimmune arthritis by targeting STAT3 signaling and suppressing Th17 cell responses

• Systemic infusion of SSU72 reduces joint destruction, serum immunoglobulin levels, and osteoclastogenesis in experimental arthritis

• SSU72 increases regulatory B cells (marginal zone B cells and B10 cells)

• SSU72 is essential for peripheral Treg differentiation and prevention of spontaneous inflammation

Experimental approaches to investigate SSU72 in autoimmunity:

• In vitro studies:

  • Analyze effects of SSU72 overexpression or knockdown on T cell differentiation into Th17 vs. Treg subsets

  • Measure cytokine production (IL-17, IFNγ, IL-4) in SSU72-modified T cells

  • Assess STAT3 phosphorylation status in response to SSU72 modulation

• In vivo models:

  • Use conditional knockout mice (CD4-Cre SSU72fl/fl) to assess T cell-specific effects

  • Implement experimental autoimmune arthritis models with SSU72 treatment

  • Monitor development of spontaneous inflammation in SSU72-deficient mice

• Clinical correlations:

  • Analyze SSU72 expression in patient samples with autoimmune conditions

  • Investigate correlation between SSU72 downregulation and mucosal tolerance defects

  • Evaluate genetic polymorphisms in SSU72 in autoimmune disease cohorts

The research indicates that targeting SSU72 activity could be a promising therapeutic approach for autoimmune diseases by balancing inflammatory and regulatory immune responses.

How does SSU72 contribute to telomere maintenance and DNA replication?

SSU72 plays an essential role in telomere biology and genome stability:

• Telomere replication termination:

  • SSU72 phosphatase functions as a conserved telomere replication terminator

  • It is responsible for terminating the cycle of telomere replication in fission yeast

• Telomere stability maintenance:

  • SSU72 downregulation in human cells (HT1080) results in higher levels of multiple telomere signals (MTS)

  • MTS is an indicator of telomere fragility and replication stress

  • SSU72 knockdown causes levels of MTS comparable to those induced by aphidicolin (a DNA polymerase inhibitor)

• DNA damage response at telomeres:

  • SSU72 downregulation results in telomere-induced foci (TIF), measured by 53BP1 localization to telomeres

  • This phenotype occurs in multiple cell lines (HT1080 and HeLa)

• Experimental approaches to study SSU72 in telomere maintenance:

  • Metaphase spread analysis to measure multiple telomere signals (MTS)

  • TIF assays to assess DNA damage at telomeres

  • shRNA-mediated knockdown of SSU72 followed by telomere functionality assessment

  • Combined treatments with replication stress inducers (e.g., aphidicolin)

These findings establish SSU72 as a critical factor in telomere biology with implications for understanding cellular aging, cancer development, and genomic instability mechanisms.

What is the relationship between SSU72 and liver function, particularly in hepatocellular carcinoma development?

SSU72 serves as a critical regulator of liver function and hepatocellular carcinoma (HCC) development:

• Tumor suppressor function:

  • Loss of Ssu72 leads to marked susceptibility to HCC development in various chemical and metabolic syndrome-induced models

  • Ssu72-deleted livers show dramatic increases in weight, number, and size of tumor foci compared to wild-type mice

  • Ssu72 deletion accelerates tumor formation, with dysplastic nodules, adenoma, and HCC appearing within 4 months of diethylnitrosamine (DEN) administration

• Hepatic progenitor cell regulation:

  • Ssu72 depletion results in a remarkable increase in proliferating oval-shaped cells in pericentral zones upon DEN challenge

  • In response to liver damage, Ssu72-deficient livers show dramatically increased Epcam+CD11b− progenitor cells

  • Sox9+ and Epcam+ cells are strongly induced in metabolic syndrome-challenged Ssu72-deficient livers

• Hepatocyte dedifferentiation:

  • Ssu72 expression level is closely associated with cell fate of hepatocytes following liver damage

  • Downregulation of Ssu72 potentiates the induction of hepatocyte dedifferentiation

  • GFP+ and Sox9+ progenitor-like cells derived from mature hepatocytes are markedly increased in Ssu72-depleted liver

• Experimental approaches:

  • Use of liver-specific knockout models (Ssu72Δhep mice)

  • Chemical induction of HCC using diethylnitrosamine (DEN)

  • Metabolic syndrome models (STAM) combining streptozotocin and high-fat diet

  • Lineage tracing using GFP reporter systems to track cell fate

  • Flow cytometry analysis of progenitor markers (Epcam, Sox9, CD13, CD133)

These findings suggest that Ssu72 functions as a hepatic tumor suppressor by regulating hepatocyte differentiation status and progenitor cell expansion in response to injury.

How is SSU72 involved in thermogenic adaptation, and what methods can be used to study this function?

SSU72 plays a crucial role in thermogenic adaptation in brown adipose tissue (BAT):

• Thermogenic response regulation:

  • Adipocyte-specific Ssu72 knockout (aKO) mice show extreme cold sensitivity

  • When exposed to 4°C, Ssu72 aKO mice experience rapid body temperature drop (below 30°C in <6 hours)

  • Fatal hypothermia occurs after 6 hours of cold exposure in knockout mice

• Molecular mechanisms:

  • SSU72 is essential for mRNA translation of genes required for thermogenesis in BAT

  • Under cold exposure, thermogenic gene expression is significantly decreased in Ssu72 aKO BAT

  • UCP1 protein (key thermogenic protein) expression is reduced in Ssu72 aKO mice

  • SSU72 appears linked to fatty acid oxidation pathways and β3-adrenergic receptor signaling

• Experimental approaches for investigating SSU72 in thermogenesis:

  a) In vivo methods:

  • Generate adipocyte-specific knockout models using Cre-lox technology

  • Measure core body temperature during cold challenge (4°C exposure)

  • Analyze BAT histology for morphological differences

  • Perform metabolic cage studies to assess energy expenditure

  b) Molecular analyses:

  • RT-qPCR for thermogenic gene expression (UCP1, PGC1α, PRDM16)

  • Western blotting for protein levels of thermogenic markers

  • RNA-seq to identify global transcriptional changes

  • Polysome profiling to assess mRNA translation efficiency

  • Analyze fatty acid oxidation-related gene expression

  c) Mechanistic studies:

  • β3-adrenergic receptor activation experiments

  • Mitochondrial respiration assays with isolated BAT mitochondria

  • In vitro differentiation of brown adipocytes with SSU72 modulation

These approaches can help elucidate how SSU72 regulates thermogenesis with implications for metabolic disorders and obesity research.

What advanced techniques are most effective for studying SSU72 protein-protein interactions?

Several advanced techniques can effectively investigate SSU72 protein-protein interactions:

• Affinity purification-mass spectrometry (AP-MS):

  • Successfully used to demonstrate specific interaction between SSU72 and ZAP-70 in T cells

  • Enables unbiased identification of novel interaction partners

  • Can be coupled with SILAC for quantitative analysis of dynamic interactions

• Co-immunoprecipitation (Co-IP):

  • Effective for confirming direct interactions between SSU72 and partners like ZAP-70

  • Can verify complex formation between SSU72 and PLCγ1

  • Should include appropriate controls (IgG control, reverse IP)

• Proximity-dependent labeling:

  • BioID or TurboID fusion proteins to identify proteins in close proximity to SSU72

  • APEX2 labeling for spatial-temporal resolution of interactions

  • Particularly useful for capturing transient or weak interactions

• Förster Resonance Energy Transfer (FRET):

  • For studying interactions in living cells

  • Can detect SSU72 interactions at different subcellular locations (membrane, cytoplasm, nucleus)

  • Useful for measuring dynamic changes in protein interactions following stimuli

• Protein complementation assays:

  • Split-luciferase complementation assay

  • Bimolecular Fluorescence Complementation (BiFC)

  • Allows visualization of interaction sites within the cell

• Surface Plasmon Resonance (SPR):

  • For measuring binding kinetics and affinity constants

  • Requires purified recombinant proteins

  • Can determine if interactions are direct or require additional factors

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps interaction interfaces between SSU72 and binding partners

  • Provides structural insights into how binding occurs

  • Useful for designing inhibitors or activators of specific interactions

• Crosslinking Mass Spectrometry (XL-MS):

  • Captures transient interactions via chemical crosslinking

  • Identifies specific amino acids involved in interactions

  • Compatible with complex protein mixtures

For SSU72 specifically, choose techniques based on:

• The cellular context (T cells, liver, BAT)

• The stimulation conditions (TCR activation, metabolic challenge)

• The subcellular compartment of interest (membrane, cytoplasm, nucleus)

• The expected strength and duration of the interaction

For example, to study dynamic TCR-induced interactions, FRET or proximity labeling in live T cells would be most informative, while AP-MS might be better for identifying the complete SSU72 interactome.