FIG. 5.

IKKβ and βTrCP1 trigger serine 927-dependent degradation of p105. (A) Transfections were performed in quadruplicate. One fourth of the CaPO₄ DNA precipitate was used to transfect a 6-cm plate of 293 cells. Each transfection contained a total of 20 μg of DNA with 2 μg of p105 expression vector (lanes 1 to 12) or 2 μg of p105AAA (lanes 13 to 24). For cotransfection, 4 μg of IKKβ (lanes 5 to 12 and 17 to 24) and 2 μg of βTrCP1-Flag (lanes 9 to 12 and 21 to 24) were used. Cells were treated with cycloheximide (CHX) for the indicated times, and extracts were prepared in RIPA buffer containing the complete protease inhibitor cocktail (Boehringer). (B) 293 cells were cotransfected with IKKβ, βTrCP1, and wild-type p105 or various p105 proteins (residues 18 to 968) carrying point mutations as shown in Fig. 2 or with an expression vector encoding p100. Transfection, stimulation, and lysis of cells were performed as described for panel A.