FIGURE 3.

MDP treatment induces proteasomal degradation of Nod2 and Rip2. A and C, upper, RAW264.7 cells were pretreated with IFN-γ for 6 h and then stimulated with MDP for the indicated times. A, the expression of Nod2 was examined by quantitative real time PCR. Data were normalized to the expression of the β-actin gene. Data represent the mean ± S.D. of triplicates. B, cells were pretreated with IFN-γ for 6 h and then stimulated with MDP for the indicated times in the presence or absence of MG-132 (10 μm). Cell extracts were subjected to Western blot analysis for Nod2, Rip2, and actin. *: slowly migrating Nod2. C, upper, cell extracts were immunoprecipitated (IP) with anti-Nod2 antibody. The level of ubiquitination on Nod2 was analyzed. C, lower, HEK293T cells were transfected with FLAG-Nod2 and HA-WT-Ub plasmid vectors. Forty-eight hours after transfection, cells were treated with MG-132 (10 μm) for 2 h. Cell extracts were immunoprecipitated with anti-Nod2 antibody. The level of Nod2 ubiquitination was analyzed. Error bars represent the mean ± S.D. of triplicates. Data are representative of three independent experiments with similar results. L.E., long exposure; n.s., nonspecific.