Figure 5. NFI-C interaction with Smurf1 requires activation of the MAPK pathway by TGF-β signaling.

(A) MDPC-23 cells were treated with TGF-β1 (10 ng/ml) for 1 hr and then lysed. The NFI-C immunoprecipitates or whole cell lysates (WCL) were subjected to western blot analysis with the anti-Smurf1 or anti-NFI-C antibody. GAPDH was used as a loading control. (B) HEK293T cells were co-transfected with HA-tagged NFI-C and FLAG-tagged Smurf1 expression vectors for 48 hr. WCL and NFI-C immunoprecipitates were analyzed by western blot with anti-FLAG or anti-HA antibody. (C) MDPC-23 cells were incubated with TGF-β1 (1 hr, 10 ng/ml) in the presence or absence of the MEK inhibitor, U0126 (10 µM), added 1 hr prior to TGF-β1 addition. Cells were lysed, and NFI-C, p-ERK, and GAPDH levels were analyzed by western blot. (D) MDPC-23 cells were stimulated with TGF-β1 (10 ng/ml) for 1 hr in the presence or absence of the MEK inhibitor, U0126 (10 µM). WCL and NFI-C immunoprecipitates were analyzed by western blot. (E) WCL and anti-phospho-Ser/Thr-Pro immunoprecipitates were analyzed by western blot with anti-NFI-C antibody. (F) HA-tagged NFI-C protein was metabolically labeled with [γ-³²P]-ATP in HEK293T cells. Bound Smurf1 proteins were eluted from the beads and detected by western blot analysis with the indicated antibody. The incorporation of ³²P was detected by autoradiography, and the amount of HA-NFI-C was detected by western blot analysis.