Figure 4.

Activation of the ERK and NF-κB signaling pathways in THP-1 cells stimulated by soluble PrP^C-Fc treatment. (A) THP-1 cells were treated with soluble PrP^C-Fc or control Fc at the indicated concentrations for 15 min. For inhibition of ERK signaling, PD98059 (20µM) was added to the THP-1 culture 1 h before soluble PrP^C-Fc treatment. Western blotting was performed with cell lysates to detect phosphorylated ERK-1/2 and total ERK-1/2. (B) THP-1 cells were treated with soluble PrP^C-Fc or control Fc at the indicated concentrations for 15 min. Western blotting was performed with cell lysates to detect phosphorylated IKK, total IKK, phosphorylated IκB, and total IκB. (C) THP-1 Blue™ cells, which secret SEAP upon NF-κB activation, were treated with soluble PrP^C-Fc, heat-denatured PrP^C-Fc, or control Fc at the indicated concentrations for 48 h. Secreted SEAP in the culture supernatant was quantified by a colorimetric assay as described in Materials and Methods, and the OD at 655 nm was measured with a microplate reader. The assay was performed in triplicate. Bar graphs represent the mean±SEM. Statistical analysis was performed in comparison with untreated control. ^*p<0.05; ^**p<0.01.