Figure 3.

Activation of the UPR by C282Y HFE. (A) HEK 293 cells were transfected with pcDNA3.1 (empty vector, EV), WT HFE, or C282Y HFE (M) expression plasmids and a grp78-promoter luciferase reporter gene vector (grp78-luc) with or without pZeoSv2+ (data not shown), pMA1AT, or pZA1AT. As positive control or treatment, five hours post transfection cells were incubated with 2.5 μM thapsigargin or 300 μM TUDCA acid for 24 h. Cells were incubated for 24 h in serum-complete medium. Luciferase production in cell lysates was measured by luminometry. Levels are expressed as light units (LU) per microgram of total protein. C282Y HFE activated grp78-luc expression (**, p = 0.0011) compared to WT HFE. Treatment with TUDCA significantly decreased grp78-LUC expression (##, p = 0.0006) compared to WT HFE. Z A1AT transfection significantly increased grp78-LUC expression (*, p = 0.016) compared to N HFE. Assays were performed in triplicate and are representative of at least three separate experiments. (B) GRP78/BIP protein production was analyzed by western blot in cytosolic extracts (10 μg) from HEK293 cells transfected with pcDNA3.1 (lane 1), WT HFE (lane 2), C282Y HFE (lane 3) or HEK293 cells stimulated with 2.5 μM thapsigargin (lane 4; n = 3). (C) GRP78/BIP protein production were analyzed by western blot in cytosolic extracts (10 μg) from HEK293 cells transfected with C282Y HFE untreated (lane 1) treated with 200 μM TUDCA (lane 2) 300 μM TUDCA (lane 3). (D) GRP78/BIP protein production were analyzed by western blot in cytosolic extracts (10 μg) from HEK293 cells transfected with pZA1AT with pcDNA3.1 (lane 1), WT HFE (lane 2) or C282Y HFE (lane 3) or HEK293 cells stimulated with 2.5 μM thapsigargin (lane 4; n = 3). Blots were stripped and probed for β-Actin to confirm equal loading.

Lawless et al. BMC Cell Biology 2007 8:30   doi:10.1186/1471-2121-8-30
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