Figure 5
PD increases LDLR half-life by blocking LXR-induced IDOL expression. (A, B) HepG2 cells were treated with 10 μM T0901317 with or without 2.5 μM PD for 24 h. RT-PCR and Real-time PCR assay were performed to measure the mRNA levels of IDOL and GAPDH loading control. The IDOL mRNA expression levels represent the mean ± SD from three independent experiments. *P < 0.05 by one-way ANOVA with Tukey’s post hoc test. (C) HepG2 cells were treated with the indicated concentration of PD for 24 h. Western blot analysis was performed to determine the protein levels of LXRα and GAPDH from the cell lysates. (D) HepG2 cells were treated with either DMSO or 2.5 μM PD for 24 h and then added with 100 μg/mL CHX for the indicated time. LDLR protein was detected by western blotting and the intensity of LDLR protein was determined using Image J software and normalized to that of GAPDH control. (E) HepG2 cells were transfected with HA-ubiquitin plasmids (1 μg). The cells were treated with 2.5 μM PD or 10 μM T0901317 for 18 h and 50 nM Baf A1 for 6 h before being lysed. Cell lysates were immunoprecipitated with anti-LDLR and ubiquitinated LDLR was analyzed by immunoblotting as indicated.
