Application of STUPPIT to identify novel intermediary proteins between SMAD4 and β-catenin, central hubs of BMP and Wnt pathway.
<p><b>(A)</b> Schematic of the opposing effects of BMP and Wnt signaling on stem cell behavior. <b>(B)</b> STUPPIT strategy for labeling proteins that bridge SMAD4 and β-catenin. <b>(C)</b> STUPPIT workflow for capturing intermediary proteins between SMAD4 a...
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2025
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| Gaia: | <p><b>(A)</b> Schematic of the opposing effects of BMP and Wnt signaling on stem cell behavior. <b>(B)</b> STUPPIT strategy for labeling proteins that bridge SMAD4 and β-catenin. <b>(C)</b> STUPPIT workflow for capturing intermediary proteins between SMAD4 and β-catenin. 3 × Flag-TbN-PupE stably expressed cells were co-transfected with SMAD4-PafA-Myc&HA-TbC or SMAD4-PafA-Myc&HA-TbC-β-catenin. STUPPIT labeled intermediary proteins between SMAD4 and β-catenin were enriched by streptavidin pulldown and analyzed by mass spectrometry. Samples 1–6 (SMAD4&TbC) and 7–12 (SMAD4&TbC-β-catenin) were each pooled into three groups (1 + 2, 3 + 4, 5 + 6) and (7 + 8, 9 + 10, 11 + 12) to yield three replicates per condition. <b>(D)</b> Volcano plots of the potential intermediary proteins between SMAD4 and β-catenin enriched by STUPPIT approach I after mass spectrometry analysis. Proteins with unique peptides ≥10 in the SMAD4&TbC-β-catenin over SMAD4&TbC control group were first filtered. Proteins with fold change (SMAD4&TbC-β-catenin over SMAD4&TbC) > 1.5 were labeled as red dots in the volcano plots. <b>(E)</b> Heatmap of the unique peptide counts for the indicated candidates in SMAD4&β-catenin and SMAD4&control groups identified by mass spectrometry analysis. Underlying mass spectrometry data can be found in the ProteomeXchange (PXD069286).</p> |
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