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. 2021 Jan 4;283(6):3272–3280. doi: 10.1074/jbc.M708033200

FIGURE 3.

FIGURE 3

N protein interacts with Smad3.A, N protein interacts with Smad3. HEK293T cells were co-transfected with pEBG1-GST-N (2 μg) and FLAG-tagged Smad plasmids (5 μg each) as indicated. Cell lysates were incubated with Sepharose 4B-glutathione beads, and GST-N protein associated Smads were revealed by anti-FLAG immunoblotting (upper panel). The protein expression was confirmed with immunoblotting of total cell lysates (middle and lower panels). B, N protein interacts with endogenous Smad3 in HPL1 cells. After HPL1 cells were transfected with pEBG1 or pEBG1-GST-N for 40 h, the cells were harvested for GST pulldown. GST-N-associated Smad3 proteins (upper panel) and total protein expression (middle and lower panels) were revealed by immunoblotting. C, N protein interacts with Smad3 mutants. HEK293T cells were co-transfected with pEBG1-GST-N (2 μg) and FLAG-tagged Smad3 wild-type (WT) or mutant plasmids (5 μg each) as indicated. Cell lysates were incubated with Sepharose 4B-glutathione beads and GST-N protein-associated Smad3 were revealed by anti-FLAG immunoblotting (upper panel). The protein expression was confirmed with immunoblotting of total cell lysates (middle and lower panels). D, N protein interacts with the Smad3 MH2 domain. HEK293T cells were co-transfected with pEBG1-GST-N (2 μg) and pCMV5-HA-Smad3, MH1 (aa 2–132), MH1 plus linker (aa 2–225), and MH2 (aa 226–425) (5 μg each) as indicted. GST pulldown assay was performed similarly as in A. GST-N protein associated Smad3 was revealed by anti-HA immunoblotting (upper panel). Protein expression was confirmed immunoblotting (middle and lower panels). E, Smad3 interacts with both of the N-terminal and C-terminal domains of N protein. HEK293T cells were co-transfected with N protein and its deletion mutant constructs (5 μg each) as indicted. Cell lysates were incubated with Sepharose 4B-glutathione beads and purified bacteria-expressed GST or GST-Smad3 protein. GST-Smad3-associated N proteins (upper panel) and protein expression (middle and lower panels) were revealed by immunoblotting. F, the C-terminal domain of N protein is important to enhance TGF-β-induced expression of CAGA-luciferase. HPL1 cells were co-transfected with CAGA-luciferase reporter (0.5 μg) and pCMV5 empty vector or pCMV5-HA-N, -N (2–210), -N (211–422), -N (232–422) (0.5 μg each) as indicated. Luciferase activity was determined as in Fig. 1A. The asterisks indicate a statistically significant difference (**, p < 0.01). RLU: relative luciferase units.