Figure 4: Localization of Prohibitins in Schwann cells.

(a) Schwann cell pseudopods contain some mitochondria. Mitotracker staining (red) in Schwann cells on the top (cell bodies) or bottom (pseudopods) of the chamber, after induction with neuronal membranes. Cells were co-stained with FITC-phalloidin and DAPI. The white box is enlarged in the right panel. Arrowheads indicate mitochondria in pseudopods. (b) Western blots of protein lysates from Schwann cell pseudopods and cell bodies, after induction by neuronal membranes. Equal amount of proteins were separated by SDS/PAGE, probed for Prohibitin-1 and Prohibitin-2 and stained with amido black. Prohibitin-2 was enriched in the pseudopod fraction. Error bars indicate s.d. n=3 independent experiments. Statistical analyses were performed using t-test. *P< 0.05. (c) Immunolocalization of Prohibitin-2 in cultured Schwann cells. Cells were stained for Prohibitin-2 (green) and Mitotracker (red). Prohibitin-2 is detected mostly in mitochondria. (d) Immunolocalization of Prohibitin-2 in Schwann cell’s pseudopods, on the bottom of the Boyden chamber, after induction with neuronal membranes. Cells were stained for Prohibitin-2 (green), Mitotracker (red) and β1-Integrin (cyan). Prohibitin-2 is found in pseudopods, where it partially co-localizes with mitotracker and in some areas co-localizes with β1-Integrin. Arrows show areas where Prohibitin-2 co-localizes with β1-Integrin, but not mitotracker. The white box is enlarged in the bottom panels, and shows a Schwann cell lamellipodia-like protrusion stained for β1 integrin and Prohibitin-2, but not mitotracker (arrowheads). (e) Streptavidin-pull-down of biotinylated cell-surface proteins in Schwann cells. Equal amount of proteins were pulled-down by Streptavidin–Sepharose, separated by SDS/PAGE and probed for Prohibitin-2 and Pdha1 (mitochondrial marker) or Prohibitin-2 and β1-Integrin (plasma membrane marker). Prohibitin-2 and β1-Integrin, but not Pdha1, are present in the biotin fraction. Probing for β1-Integrin and Pdha1 were performed on two different blots. (f) Immunoprecipitation of Prohibitin-2 in Schwann cells after biotinylation of cell-surface proteins. Equal amount of proteins were precipitated by Prohibitin-2 antibody, separated by SDS/PAGE and stained for Streptavidin-HRP then Prohibitin-2. A biotionylated protein at the size of Prohibitin-2 is present (35 kDa, arrow). Other biotinylated proteins co-immunoprecipitate with Prohibitin-2. The band in the unbound fraction (asterisk) corresponds to the Streptavidin–HRP detection performed first. (IP: immunoprecipitation, Ub: unbound fraction). Scale bars, 40 μm (a,c); 10 μm (d).