FIGURE 6.

Fn14 was degraded by the ubiquitin-proteasome system. A, MC3T3-E1 cells were stimulated with cyclic mechanical stretch (12%) for 6 h. After stretch stimulation, cells were incubated with CHX (5 μg/ml) in the presence of dimethyl sulfoxide (DMSO) or MG132 (10 μm) for the indicated periods. The cell lysates were subjected to immunoblotting (IB) using the indicated antibodies. B, schematic of mouse Fn14^WT, Fn14^K48R, and Fn14^K109R. All expression plasmids were FLAG-tagged at their C termini. C, CHX chase analysis of Fn14 mutants. MC3T3-E1 cells were transfected with the indicated expression plasmids. After 24 h, cells were treated with CHX for the indicated periods. Then, the cell lysates were subjected to immunoblotting using the indicated antibodies. O/N indicates overnight. D, in vivo ubiquitination (Ub) assay of Fn14^WT and Fn14^K109R. See “Experimental Procedures” for details. IP, immunoprecipitation. E, MC3T3-E1 cells were transfected with the indicated expression plasmids. After 24 h, cells were lysed and analyzed by immunoblotting with the indicated antibodies. A, C, D, and E, the data represent one of at least three experiments.