JCS021816 Supplementary Material

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• Supplemental Tables 1-6 -

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• Supplemental Figure S1 -

  Fig. S1. A stack of 1 µm optical slices ascending from the plane of substrate attachment, of sensory neurons and substrate cells, immunolabelled for PrP^C (Alexa-Fluor-594-coupled antibody, red) and DAPI labelling chromatin (blue). The three PrP^C-expressing neurons are labelled 1, 2 and 3, and, in the lower layers, the DAPI-labelled nuclei of substrate cells asterisks in the base layer; these are mostly satellite cells; Schwann cells begin to divide and ensheath the axons, and fibroblasts slowly accumulate with prolonged (2-6 week) cultures. Axons, with punctate PrP^C immunolabelling, can be seen at the lower levels. The top image is towards the middle of neurons 1 and 2, and near the top of the smaller neuron 3. Each image shown comprises 10 serial pictures taken at intervals of 100 nm in the z-axis using a piezoelectric drive on the 63× objective on a Zeiss Axiovert microscope. The entire stack (plus seven extra flanking sections at each end of the stack that were discarded after image deconvolution) were deconvolved by Volocity software and reassembled into the images shown. Bar (top section), 5 µm.

• Supplemental Figure S2 -

  Fig. S2. Heat-shock has no effect upon the surface or internal levels of PrP^C. (A,B) Surface PrP^C immunolabelling of sensory neurons after 4 hours at 37°C (A) or 39°C (B). (C-F) Immunolabelling of total PrP (red) and BiP (green) in sensory neurons placed for 4 hours at 37°C (C,E) and 39°C (D,F) before being fixed, permeabilised and immunolabelled as above. Bar, 5 µm.