Fig 1.
Lipid spot blots identify N1 fragment binding to PS and PA.
A. Diagram showing full length PrP and the regions comprising the N1 and N2 cleavage fragments. B. Schematic indicating the lipid spot arrangement on the membrane. C. PrP23-111 (N1) incubation with the lipid spot blots at pH 7 and pH 5 with and without pre-loading with four molar equivalents CuCl2 followed by western blotting with SAF32 antibody (directed against amino acids 51–89). D. Densitometric quantification of spot intensity, n = 3, significance over blank control is shown as *p<0.05, ***p<0.001.
Fig 2.
N2 binds PS and PA lipid spots.
A. Schematic indicating the lipid spot arrangement on the membrane. B. PrP23-89 (N2) incubation with the lipid spot blots at pH 7 and pH 5 with and without pre-loading with four molar equivalents CuCl2 followed by western blotting with SAF32 antibody (directed against amino acids 51–89). C. Densitometric quantification of spot intensity, n = 3, significance over blank control and between conditions is shown as *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Lipid binding specificity is determined by regions of N1 and N2.
Lipid spot blots (incubated at pH 7) of peptides corresponding to regions of N1 and N2, including residues 23–50 (A), residues 51–89 comprising the copper-binding region and therefore tested with and without copper saturation (B) and an N1 fragment lacking the residues of the copper-binding region, Δ51–89 (C). For 23–50 and N1Δ51–89, copper saturation was not tested as neither fragment contains the octarepeat copper-binding domain and blotting used the N-terminally targeted 8B4 antibody as SAF32 targets residues 79–92. D. Lipid spot blots of a mutant N2 P26/28A fragment with and without copper saturation at pH 7 detected with SAF32. E. Densitometric quantification of spot intensity for the domains of N1 and N2, n = 1. F. Densitometric quantification of spot intensity of the P26/28A mutation of N2 with and without copper saturation. Apo N2 intensities are shown for comparison of differences between the mutated and wild-type (WT) sequence, n = 3. Significance over blank control is shown in black and significant differences in detection from the wild type sequence of N2 are shown in red, *p<0.05, ***p<0.001.
Fig 4.
Serum deprivation causes changes to PS and PA in CF10 cells.
A. Laurdan GP changes following serum deprivation for 30 minutes as compared with benzyl alcohol (BA) and filipin III controls. n = 3. B. Live cell imaging of NBD-PS labelled CF10 cells. Image intensity is thresholds have been selected to view detail in the staining pattern and do not represent a comparison of fluorescence intensity. Scale bars = 20 μm. C. Fluorescence emission spectra of NBD-PS labelled cells following transfer into serum-free medium, scans were taken immediately after media replacement. D. Anisotropy of NBD-PS in CF10 cells with and without serum present. n = 3. E. Counts from magnetic separation of cells that have lost membrane asymmetry allowing them to bind PS at 5 and 15 minutes post serum withdrawal. n = 4. F. ROS production detected by DCF fluorescence when cells are serum-starved and with exposure to butan-1-ol to inhibit PLD activity. n = 4. G. Measurement of cellular phosphatidic acid concentration 30 minutes after commencing serum deprivation. n = 3. H. Measurement of phospholipase-D activity following 15 minutes serum starvation. n = 3. For all panels, *p<0.05, ***p<0.001.
Fig 5.
N2, but not N1, reverses PS externalisation and PA increase in the absence of further membrane changes.
A. Laurdan GP of serum starved cells alone and when treated N2 (23–89), N1 (23–111), N2 with the two prolines within the N-terminal polybasic region mutated to alanines (23-89P26/28A, the octarepeat region (51–89), copper-saturated (four molar equivalents) N1, N2, 23-89P26/28A and 51–89, 23–50 and equivalent copper without peptide, measured under the same conditions used in Fig 4. n = 3. B. Annexin V magnetic separation of N1 and N2 with and without copper saturation. Filled bar indicates 10% (v/v) serum and hollow bars show conditions with 0% serum. n = 3. C. NBD-PS anisotropy of N2 with and without copper saturation. n = 3. D. Phospholipase-D activity within N1 and N2 (+/- copper), and 23–50 (no copper) serum starved cells. n = 3. E. Relative PA concentrations within N2 treated (+/- copper) serum starved cells. n = 3. Fig *p<0.05.