FIGURE 4. Smurf1 or Smurf2 siRNA blocks TNF-induced Runx2 degradation.

2T3 cells were infected with retroviral supernatant containing Smurf1 and/or Smurf2 siRNAs or empty vector, then cells were transfected with F-Runx2 expression plasmid (4 μg/dish) and treated with 7.5 ng/ml TNF for 72 h. Smurf1 (A) and Smurf2 (B) mRNA levels were measured by real time RT-PCR. The values are the mean ± S.E. of three dishes. The -fold increase was calculated as described in Fig. 2A. *, p<0.05 versus the empty vector-infected TNF group. C, the expression of F-Runx2 was determined by Western blot analysis using anti-FLAG antibody as described in Fig. 2E. D, ALP mRNA expression was assessed by real time RT-PCR. The values are the mean ± S.E. of three dishes. The -fold increase was calculated as described in Fig. 2A. *, p<0.05 versus empty vector-infected TNF group.