Phospho-UBTF (Ser484) Antibody

What is UBTF and what is the significance of its Ser484 phosphorylation?

UBTF (Nucleolar Transcription Factor 1) is a critical protein that recognizes ribosomal RNA gene promoters and activates transcription mediated by RNA polymerase I through cooperative interactions with the transcription factor SL1/TIF-IB complex. It binds specifically to the upstream control element and can activate Pol I promoter escape . Phosphorylation at Ser484 by G1-specific cyclin-dependent kinase (cdk)/cyclin complexes plays a key role in cell cycle-dependent regulation of rRNA synthesis and correlates with activation of rDNA transcription during G1 progression . This post-translational modification represents an important regulatory mechanism for modulating the assembly of the transcription initiation complex in a proliferation- and cell cycle-dependent manner .

What applications are Phospho-UBTF (Ser484) antibodies suitable for?

Phospho-UBTF (Ser484) antibodies are primarily suitable for Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and ELISA applications . For optimal results, recommended dilutions typically range from 1:500-1:2000 for WB, 1:50-1:300 for IHC, 1:50-200 for IF, and 1:5000-1:10000 for ELISA applications . The antibody specifically detects endogenous levels of UBTF protein only when phosphorylated at Ser484, making it valuable for investigating phosphorylation-dependent cellular processes .

What experimental controls should be included when using Phospho-UBTF (Ser484) antibody?

When using Phospho-UBTF (Ser484) antibody, several controls should be implemented to ensure experimental validity:

• Phosphatase treatment control: Treating a sample with lambda phosphatase before antibody application should eliminate signal, confirming phospho-specificity.

• Cell cycle synchronization: Since Ser484 phosphorylation occurs during G1 phase, comparing synchronized cells at different cell cycle stages provides validation of signal specificity .

• Isotype controls: Using appropriate rabbit IgG isotype controls helps distinguish specific from non-specific binding .

• Secondary antibody controls: Testing secondary antibodies alone ensures the absence of non-specific signals.

• UBTF knockdown/knockout: Using shRNA for UBTF gene silencing, as described in research protocols, provides an essential negative control for antibody specificity .

How can researchers distinguish between phosphorylation at Ser484 and other UBTF phosphorylation sites?

Distinguishing between different UBTF phosphorylation sites requires careful methodological approaches:

• Site-specific antibodies: Using antibodies that specifically recognize phospho-Ser484 versus other sites (e.g., Ser388) .

• Tryptic phosphopeptide mapping: This technique can differentiate between various phosphorylation sites on UBTF. Research has identified distinct tryptic peptides containing phosphorylated residues, with peptide "a" (aa 481-486) containing phospho-Ser484 .

• Mutational analysis: Comparing wild-type UBTF with site-specific mutants (e.g., S484G or S484D) in functional assays can confirm site-specific effects .

• Mass spectrometry: For unambiguous identification, phosphopeptides can be analyzed by mass spectrometry to precisely map phosphorylation sites.

• Sequential immunoprecipitation: Using antibodies against different phosphorylation sites sequentially can help determine if multiple sites are phosphorylated simultaneously.

What are optimal protocols for monitoring UBTF Ser484 phosphorylation changes during cell cycle progression?

For monitoring UBTF Ser484 phosphorylation across the cell cycle:

• Cell synchronization methods:

  • Serum starvation-release: Synchronize cells in G0/G1 by serum starvation, then release by adding serum

  • Thymidine block: Use for S-phase synchronization

  • Nocodazole treatment: For M-phase arrest

• Time-course analysis protocol:

  • Harvest cells at regular intervals after synchronization (e.g., every 2-4 hours)

  • Prepare nuclear/nucleolar fractions using subcellular protein fractionation

  • Perform Western blot analysis using Phospho-UBTF (Ser484) antibody (1:500-1:1000 dilution)

  • Include cell cycle markers (e.g., cyclin D, cyclin E) as parallel controls

  • Quantify relative phosphorylation levels by densitometry

• Flow cytometry co-analysis:

  • Fix cells in 70% ethanol

  • Perform dual staining with Phospho-UBTF (Ser484) antibody and propidium iodide

  • Analyze correlation between phosphorylation signal and cell cycle position

How does UBTF Ser484 phosphorylation interact with other post-translational modifications?

Research indicates complex interactions between different UBTF post-translational modifications:

This interplay suggests a coordinated regulation mechanism where modifications at different sites work together to modulate UBTF activity throughout the cell cycle.

What are common challenges when working with Phospho-UBTF (Ser484) antibody in Western blot applications?

Several technical challenges may arise when detecting phospho-UBTF (Ser484):

• Phosphatase activity during sample preparation:

  • Solution: Add phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate) to all buffers

  • Maintain samples at 4°C throughout processing

  • Process samples quickly to minimize dephosphorylation

• Low signal intensity:

  • Increase antibody concentration (up to 1:500 dilution)

  • Extended primary antibody incubation (overnight at 4°C)

  • Use enhanced chemiluminescence (ECL) detection systems

  • Enrich nucleolar fraction through subcellular fractionation

• Non-specific bands:

  • Increase blocking time (5% BSA in TBST for 2 hours)

  • Optimize washing steps (at least 4×10 minutes with TBST)

  • Verify expected molecular weight (89kDa for UBTF)

  • Pre-absorb antibody with non-phosphorylated peptide

How can researchers effectively use Phospho-UBTF (Ser484) antibody to study rRNA transcription regulation?

To effectively study rRNA transcription regulation using Phospho-UBTF (Ser484) antibody:

• Correlation analysis protocol:

  • Parallel assessment of Ser484 phosphorylation and rRNA synthesis

  • Label newly synthesized RNA using 5-ethynyl uridine followed by flow cytometry analysis

  • Perform chromatin immunoprecipitation (ChIP) using Phospho-UBTF (Ser484) antibody to assess rDNA promoter occupancy

  • Correlate phosphorylation levels with transcriptional output

• Manipulation experiments:

  • Compare wild-type cells with those expressing UBTF mutants (S484G/S484D)

  • Use CDK inhibitors to block Ser484 phosphorylation

  • Study transcriptional activation in response to serum stimulation, which triggers Ser484 phosphorylation

  • Employ inducible shRNA systems for UBTF knockdown

• Co-immunoprecipitation studies:

  • Use Phospho-UBTF (Ser484) antibody to immunoprecipitate phosphorylated UBTF

  • Analyze co-precipitating factors (SL1/TIF-IB complex, RNA polymerase I)

  • Compare protein interactions between phosphorylated and non-phosphorylated forms

How can researchers use Phospho-UBTF (Ser484) antibody to investigate cancer biology?

Phospho-UBTF (Ser484) antibody can provide valuable insights into cancer research:

• Comparative analysis protocol:

  • Compare UBTF Ser484 phosphorylation levels between normal and cancer cells

  • Analyze correlation with proliferation markers

  • Evaluate changes in rRNA synthesis using 5-ethynyl uridine labeling

  • Assess phosphorylation patterns across cancer cell lines (e.g., THP-1, U2OS)

• Therapeutic response monitoring:

  • Evaluate changes in Ser484 phosphorylation after treatment with:

    • CDK inhibitors

    • Transcription inhibitors

    • Chemotherapeutic agents

  • Correlate phosphorylation changes with treatment efficacy

• Clinical specimen analysis:

  • Optimize IHC protocols (1:50-1:100 dilution) for tissue microarrays

  • Develop scoring systems based on phosphorylation intensity

  • Correlate with patient outcomes and clinicopathological features

What are the best approaches for studying the relationship between UBTF mutations and its phosphorylation?

To study the relationship between UBTF mutations and phosphorylation:

• Site-directed mutagenesis strategy:

  • Generate constructs with:

    • Phospho-mimetic mutations (S484D)

    • Phospho-null mutations (S484G)

    • Disease-associated mutations (e.g., UBTF-ITD mutants)

  • Transfect into appropriate cell models (e.g., U2OS cells)

  • Assess phosphorylation at other sites using phospho-specific antibodies

• Structural biology approaches:

  • Use molecular dynamics simulations to predict how mutations affect kinase recognition

  • Analyze crystal structures of wild-type versus mutant UBTF

  • Study conformational changes upon phosphorylation

• Functional rescue experiments:

  • Silence endogenous UBTF using shRNA

  • Rescue with wild-type or mutant constructs

  • Assess restoration of phosphorylation patterns and transcriptional activity

What are the optimal storage conditions for Phospho-UBTF (Ser484) antibody?

For optimal maintenance of antibody activity:

• Long-term storage:

  • Store at -20°C for up to 1 year from the date of receipt

  • Avoid repeated freeze-thaw cycles

  • Store in small aliquots to minimize freeze-thaw cycles

• Working solution preparation:

  • Dilute in appropriate buffer immediately before use

  • For western blot: dilute in 5% BSA/TBST solution

  • For IHC: dilute in antibody diluent with background reducing components

• Shipping and temporary storage:

  • Shipped at 4°C

  • Upon delivery, immediately aliquot and transfer to -20°C

  • Can be kept at 4°C for up to one week while in use

How should researchers validate newly received Phospho-UBTF (Ser484) antibody lots?

To ensure consistency between antibody lots:

• Positive control validation protocol:

  • Test with samples known to express phosphorylated UBTF (e.g., proliferating cells)

  • Compare signal intensity and specificity with previous lots

  • Verify molecular weight (89kDa) and band pattern

• Phosphorylation-dependence test:

  • Compare samples from serum-starved versus serum-stimulated cells

  • Treat half of a positive sample with lambda phosphatase

  • Confirm loss of signal after phosphatase treatment

• Cross-reactivity assessment:

  • Test reactivity across species (Human, Mouse, Rat)

  • Verify absence of non-specific bands

  • Compare results with published literature or reference data