QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 6. Effect of Cu²⁺ on the cell-surface distribution of PrP-N and PrP-oct. (A) Schematic of the PrP constructs used in this study. PrP^C is shown as the mature, full-length protein (residues 23-231) with its C-terminal GPI anchor, the N-terminal polybasic region (KKRP, residues 23-26, chequered box) and the octapeptide repeats (residues 51-91, shaded). PrP-oct lacks the entire octapeptide repeat region and PrP-N lacks the N-terminal polybasic region. In PrP-CTM, the GPI anchor attachment signal is replaced with the transmembrane (filled box) and cytoplasmic domains (cross-hatched) from angiotensin-converting enzyme (Walmsley et al., 2001). (B) SH-SY5Y cells expressing PrP-N were surface biotinylated and then incubated in the absence or presence of 100 µM Cu²⁺. Prior to lysis, the cells were incubated with trypsin to digest cell-surface PrP. Cells were then lysed, and total PrP was immunoprecipitated from the sample using antibody 3F4 and then subjected to western blot analysis. The biotin-labelled PrP fraction was detected with peroxidase-conjugated streptavidin. SH-SY5Y cells stably expressing (C) PrP-N or (D) PrP-oct were seeded onto glass coverslips and grown to 50% confluency. Cells were then pre-incubated with antibody 3F4 at a dilution of 1:1000 in PBS for 30 minutes at 4°C, washed three times in PBS and then incubated for 20 minutes at 37°C in OptiMEM in the absence or presence of 100 µM Cu²⁺ along with 500 µM tyrphostin A23. Where indicated, cells were incubated at 4°C for 10 minutes with PBS containing 1% Triton X-100 prior to fixation. Cells were fixed, incubated with Alexa488-conjugated secondary antibody and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Bar, 10 µm. Average fluorescent intensity per unit length of membrane for PrP-N and PrP-oct were as follows: –Cu²⁺, 11.58 and 10.54 units; +Cu²⁺, 12.61 and 10.10 units; +Cu²⁺ + Triton X-100, 9.09 and 8.81 units, respectively. (E) The percentage of the cell surface stained for PrP-N and PrP-oct was determined from the images in (C) and (D), respectively, as described in Fig. 4. Results are the mean±s.e.m. of three individual experiments, in each of which 30 cells were measured. Statistical differences (n=90), using Student's t-test, with probability values of P<0.05 were taken as significant.

Return to article