FIGURE 6.

The NPC1L1(S881L) is degraded through the ERAD pathway. A, proteasome inhibitor, MG-132, blocked the degradation of NPC1L1(S881L). 24 h after transfection, cells were treated with 100 μm CHX in the presence or absence of 10 μm MG-132. Then the cell membrane fractions were prepared at indicated time points and analyzed by Western blot with anti-EGFP antibody. C, K0 mutant on the S881L variant significantly stabilized the protein. Cells expressing the S881L variant or NPC1L1(S881L)-K0 were treated with 100 μm CHX for different time durations before harvest. Membrane fractions were prepared and analyzed by Western blot. E, dominant negative VCP blocks degradation of the S881L variant. Different amount of VCP-DN plasmids were co-transfected with the S881L variant. 24 h after transfection, the cells were treated with CHX and analyzed by Western blot. β-Actin was also probed to indicate the equal loading of lysates. B, D, and F, amount of NPC1L1 at each time point was quantified using densitometry and normalized against the protein level at time point 0. G, ubiquitination of wild-type and the S881L variant of NPC1L1. The EGFP-tagged wild-type and the S881L variant of NPC1L1 were transiently expressed in CRL-1601 cells. After 2 h of MG-132 treatment, immunoprecipitation was performed using anti-EGFP beads followed by anti-ubiquitin or anti-EGFP immunoblotting.