FIGURE 5.

Synphilin-1A decreases the ubiquitylation of SIAH-1. A, HEK293 cells were transfected with HA-SIAH-1 and increasing amounts of myc-synphilin-1A. Cells were incubated 12 h with 10 μm lactacystin. HA-SIAH-1 was immunoprecipitated (IP) with anti-HA antibody, and ubiquitylated SIAH-1 was detected by Western blot using anti-ubiquitin (Ubiq) antibody. The amount of immunoprecipitated SIAH-1 was determined using anti-SIAH-1 antibody N-15 (2nd panel). The levels of synphilin-1A (Sph-1A) were determined with anti-Myc antibody (3rd panel). B, in vitro translated SIAH-1 was incubated with UbcH5b, ubiquitin, and the other purified components of the ubiquitin system in the absence or in the presence of recombinant synphilin-1A. The levels of SIAH-1 ubiquitylation were determined by PhosphorImager analysis. The amount of synphilin-1A added was determined by Western blot using anti-synphilin-1A antibody (lower panel). BSA, bovine serum albumin.