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Nuclear accumulation of Smad1 with TNF-α stimulation and Traf2 silencing. MC3T3-E1 Entinostat were stimulated with TNF-α after transfection with Traf2 siRNA for 48 h. Localization of Smad1 with TNF-α stimulation after Traf2 silencing. MC3T3-E1 cells were stimulated with TNF-α (10 ng/ml) for 30 min after transfection with negative control siRNA or 100 nM Traf2 siRNA. Smad1 was detected by immunofluorescence staining. Left panel: negative control siRNA; right panel: Traf2 siRNA. FITC-stained Smad1 is shown; nuclei were stained with DAPI.
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Discussion
We used a yeast two-hybrid screen to demonstrate that Traf2 is a Smad4-binding protein, and this interaction between endogenous proteins was confirmed by a Ni–NTA pull-down assay and co-immunoprecipitation. Silencing of the Traf2 gene increased the BMP-2 and phospho-Smad1 levels, as well as the transcription factors in the BMP signaling pathway. Furthermore, we demonstrated that TNF-α stimulation increased the accumulation of Smad1 in the nucleus by Traf2 siRNA. This is the first study to show the interaction between Traf2 and Smad4 and Traf2 might regulate the nuclear accumulation of Smad1 in BMP signaling pathway under TNF-α stimulation.