Fig 1: TRAF4 diminished the expression of Smurf2 to modulate the osteogenesis process of MSCs. a Coomassie blue staining of the coimmunoprecipitated mixture separated by SDS-PAGE. b Coimmunoprecipitated mixtures were separated by SDS-PAGE and evaluated by western blot. Endogenous TRAF4 and Smurf2 in MSCs interacted with each other. c Immunofluorescence assays showed the colocalization of TRAF4 and Smurf2 in MSCs (green TRAF4, red Smurf2, blue DAPI). d The Smad1 and Runx2 protein levels were decreased in the Sh-TRAF4 group and increased in the Sh-Smurf2 group compared with those in the NC1 group, while compared with Sh-TRAF4, Sh-TRAF4/Sh-Smurf2 rescued the expression of Smad1 and Runx2. The Smad1 and Run2 protein levels increased in the OE-TRAF4 group and decreased in the OE-Smurf2 group compared with the NC2 group, while the expression levels of Smad1 and Runx2 in the OE-TRAF4/OE-Smurf2 group was decreased compared with those observed in the OE-TRAF4 group. e The simultaneous knockdown of TRAF4 and Smurf2 increased the ARS and ALP staining compared with that in the Sh-TRAF4 group (scale bar = 250 µm, black arrows), while the simultaneous overexpression of TRAF4 and Smurf2 decreased the ARS and ALP staining to the level observed in the NC2 group compared with OE-TRAF4 group (scale bar = 250 µm, black arrows). Data in (d) and (e) are presented as the means ± SD. *p < 0.05 (n = 3 independent experiments with three different MSC lines)