Journal: Cell & Bioscience
Article Title: Cellular prion protein and calcium ions trigger the neurotoxicity of α-synuclein aggregates
doi: 10.1186/s13578-025-01479-7
Figure Lengend Snippet: PrP C partially mediates the binding of αS species to neuronal membranes. A Representative STED microscopy images showing primary rat cortical neurons treated for 60 min with OB* and SF at 0.3 µM (monomer equivalents). Red and green fluorescence: MAP-2 and αS species detected by rabbit anti-MAP-2 and mouse monoclonal 211 Abs, respectively. The histograms show the Pearson’s and Manders' coefficients (M1 and M2) determined using the ImageJ software and JACOP plugin (n = 3). B Representative confocal microscopy images of SH-SY5Y cells treated for 10 min with 488-labelled OB* and SF at 0.3 μM (monomer equivalents). Green and red fluorescence: 488-labelled αS and PrP C detected with the mouse monoclonal Ab anti-PrP c , respectively. The histograms show the Pearson’s and Manders' coefficients (M1 and M2) determined as in ( A ) (n = 10 for OB* and n = 6 for SF). C Representative confocal microscopy images showing the immunostaining of PrP C in SH-SY5Y cells pre-transfected with control siRNA, or with PrP C siRNA. The histogram shows the semi-quantitative analysis of the PrP C -derived fluorescence (n = 3). D Western blot analysis of PrP C levels in SH-SY5Y cells pre-transfected with control siRNA or with PrP C siRNA. The histogram shows the semi-quantitative analysis of PrP C levels as the percentage relative to cells transfected with control siRNA, after normalizing each band to vinculin, taken as housekeeping (n = 3). E Representative confocal microscopy images of the apical sections of SH-SY5Y cells pre-transfected with control siRNA or with PrP C siRNA, and then treated for 10 min with OB* and SF at 0.3 μM (monomer equivalents). Red and green fluorescence: cell membranes and αS species detected by WGA and rabbit polyclonal anti-αS Ab , respectively. The histograms show the Pearson’s and Manders' coefficients (M1 and M2) determined as in ( A ); (n = 14 for OB* and n = 9 for SF in cells pre-treated with control siRNA; n = 11 for OB* and n = 10 for SF in cells pre-treated with PrP C siRNA). In all panels, experimental errors are S.E.M. Samples were analyzed by Student t test (** P < 0.01 and *** P < 0.001 relative to cells treated with OB* in panels A, B); °° P < 0.01 and °°° P < 0.001 relative to corresponding cells treated with control siRNAs in panels C, D, E
Article Snippet: After extensive washing, the membranes were incubated with 1:3000-diluted horseradish peroxidase-conjugated anti-rabbit secondary antibodies (ab6721, Abcam) for 1 h. Loading control was performed by incubating the membranes with 1:5000-diluted mouse monoclonal anti-vinculin Ab (66305-1-Ig, Proteintech).
Techniques: Binding Assay, Microscopy, Fluorescence, Software, Confocal Microscopy, Immunostaining, Transfection, Control, Derivative Assay, Western Blot
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