Fig. 6. Cellular STI1 binds recombinant PrPc. (A) HEK 239T cells transfected with GFP–STI1 or GFP, or non-transfected (NT) were incubated in the absence or presence of 20 mg of His6-PrPc followed by incubation with mouse anti-PrPc or non-immune serum and anti-mouse R-phycoerythrin conjugate. Analyses were carried out using a Becton Dickinson FACScan Cytometer. The specific fluorescence intensity was determined by subtraction of the fluorescence obtained with non-immune serum from that produced with anti PrPc serum. *P <0.01, GFP–STI1 + 20 mg PrPc versus GFP–STI1 without PrPc and #P <0.03, GFP–STI1 + 20 mg PrPc versus GFP + 20 mg PrPc, Mann– Whitney test. (B) Primary fibroblast cultures from PrP0/0 animals were incubated in the absence (b) or presence (c) of His6-PrPc followed by incubation with mouse anti-PrPc (b and c) or non-immune serum (a). (C) Whole cells (lane 1) or cellular extracts (lane 2) from PrP0/0 mice fibroblasts were incubated with His6-PrPc. Whole cells were washed, lysed and the extracts incubated with Ni-NTA–agarose. Extracts from cells without His6-PrPc addition were also incubated with Ni-NTA–agarose (lane 3). The bound material was eluted off the beads and analyzed by western blot using anti-STI1 (α-STI1, upper panel) or anti-PrPc (α-PrPc, lower panel) serum.