FIGURE 6.

Clusterin levels affect the copper export capacity of ATP7B. A, HEK293T cells were transfected with the MRE-luciferase reporter construct and co-transfected with the pEBB-FLAG empty vector, pIREShyg1/clusterin, or clusterin siRNA. Cells were either left untreated or incubated with 150 μm CuCl₂ (16 h). Luminescence was measured, and data are expressed as fold induction relative to the pEBB-FLAG empty vector. B, HEK293T cells were transfected with the MRE-luciferase reporter construct and co-transfected with pEBB/WT-ATP7B-FLAG, pEBB/WT-ATP7B-FLAG plus pIREShyg1/clusterin or pEBB/WT-ATP7B-FLAG plus clusterin siRNA. Luminescence was measured and data are expressed as fold induction relative to WT-ATP7B-FLAG. C, cell lysates following the above transfections were fractionated and immunoblotted with anti-FLAG, anti-clusterin, and anti-β-tubulin antibodies. D, densitometric analysis of ATP7B and clusterin levels expressed as relative optical density and representing the mean ± S.E. (n = 3). Asterisks indicate values that are significantly different. *, p < 0.05. E and F, cells transfected as above were left untreated or were supplemented with 150 μm CuCl₂ (16 h) and then harvested for copper analysis by inductively coupled plasma MS. Data are expressed as the mean ± S.E. (n = 4). Asterisks indicate values that are significantly different. *, p < 0.05 versus empty vector and ATP7B alone controls. KD, knockdown.