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. 2012 Jun 15;287(32):27204–27216. doi: 10.1074/jbc.M112.376616

FIGURE 5.

FIGURE 5.

Specific alteration of SMAD7, SMURF2, or SKI expression modulates T-cell TGF-β responsiveness. Hut78 T-cells were electroporated with plasmid construct containing SMAD7-FLAG or vector alone (a); nonspecific siRNA (N/S) or specific siRNA against SMAD7 (b), SMURF2 (c), or SKI (d). After 48 h, cells were stimulated via LFA-1/ICAM-1 for 6 h or unstimulated and then treated with or without 5 ng/ml TGF-β for 30 min before lysis. Cell lysates (20 μg each) were Western blotted and probed with anti-pSMAD2 or anti-SMAD2. Relative densitometric analysis of the individual pSMAD2 protein band is presented. e, untransfected or siRNA (N/S, SMAD7, SMURF2, or SKI) transfected Hut78 T-cells were stimulated via LFA-1/ICAM-1 for 6 h and then incubated with anti-CD3/CD28 for additional 24 h in the presence or absence of TGF-β. Conditioned medium was collected, and IL-2 secretion was measured by ELISA. Data are representative of three independent experiments performed in triplicate (mean ± S.E.). *, p < 0.05; NS, not significant.