Figure 3.
Endogenous and recombinant STI1 colocalize with PrPc on the surface of hippocampal neurons. A, Cultured, unpermeabilized, or permeabilized hippocampal neurons from Prnp+/+ mice were immunostained for PrPc (a, e) and STI1 (b, f). Cell nuclei were labeled with DAPI (c, g). In unpermeabilized cells, colocalization was observed at the surface of the cell soma (d). Permeabilized cells show extensive colocalization of both proteins in the cell body, perinuclear region, and neurites (h). B, Prnp+/+ primary neuronal cultures were either treated with (blue curve) or without (black curve) His6-STI1, followed by incubation with an anti-His-Tag antibody. Flow cytometry analysis detected His6-STI1 at the neuronal surface. C, Primary neuronal cultures were either treated with (STI1) or without (Control) His6-STI1. Cells were washed and lysed, and the extracts were incubated with Ni-NTA-agarose. The bound material was eluted off the beads and analyzed by immunoblotting using anti-HisTag, anti-STI1, and anti-PrPc antibodies. D, Hippocampal cultures were treated with His6-STI1-Alexa488 (a) and immunostained with anti-PrPc antibody (b). Extensive colocalization of both proteins was observed on the cell surface (c).