Fig. 3.

Inhibition of lysosomal pathway increased Niemann-Pick C1 Like 1 (NPC1L1) protein expression. A: stably transfected HuTu-80 cells were treated with bafilomycin A1 (80 nM) for 24 h. Equal amounts of total protein from control and bafilomycin A1-treated groups were resolved on SDS-PAGE and analyzed by Western blotting. B: densitometric analysis showed a significant increase in NPC1L1 protein levels. Data are presented as means ± SE from 3 different experiments. **P < 0.001 compared with control. C: HuTu-80 cells were transfected with green fluorescent protein (GFP)-tagged human (h) NPC1L1 cDNA to generate a stable cell line overexpressing NPC1L1-GFP fusion protein. Cells were treated with the proteasome inhibitor MG-132 (10 μM) and lysosome inhibitor bafilomycin A1 (80 nM) for 24 h. After 24 h, cells were harvested, and total proteins were separated by SDS-PAGE and analyzed by Western blot analysis. D: human intestinal Caco-2 cells were transiently transfected with hemagglutinin (HA)-tagged hNPC1L1. After transfection (24 h), cells were treated with proteasome or lysosome inhibitor MG-132 (10 μM) and bafilomycin A1 (80 nM), respectively, for 24 h followed by Western blot analysis using anti-HA antibody.