(a) DU145 cells were co-transfected with plasmids encoding HA-BCCIPα and LYRIC or β-galactosidase. Pairs of lanes represent two independent transfections. LYRIC overexpression resulted in decreased levels of BCCIPα protein (lanes 3&4), relative to negative control lacZ plasmid (lanes 5&6). Endogenous BCCIPβ did not appear to be affected. (b) DU145 cells were transfected with HA-BCCIPα and LYRIC or lacZ plasmids then treated with MG-132 (+) or DMSO (−)overnight. Western blot shows LYRIC overexpressed approximately 4-fold, resulting in a two-fold decrease in BCCIPα protein levels. Proteasome inhibition by MG-132 abrogated the effect. p21 was detected only with proteasome inhibition. β-actin was a loading control. (c) DU145 cells were transfected with plasmids encoding BCCIPα and full length LYRIC, deletion mutant hsNΔ169, or β-galactosidase and analyzed by Western blot. Decreased levels of HA-BCCIP were seen only with full-length LYRIC, the deletion mutant had no effect. (d) HA-BCCIP levels were determined by densitometry of the bands from Western blots as shown in panel (c). Graph depicts average and standard deviation of HA-BCCIP expression from three independent transfection experiments. The difference in HA-BCCIP protein levels in cells co-transfected with LYRIC and hsNΔ169 was statistically significant (p<0.01).