Supplementary Figure 8: Chemical structure and biochemical analysis of PTEN synthetic peptides and their permeability in neurons

a. Top panels: Chemical structure and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis for each of the peptides used for this study. Bottom panels: Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) analysis of each peptide. b. Primary hippocampal neurons (21 days in vitro) were incubated with Fluorescein-tagged PTEN-PDZ peptide (10 μM) dissolved in DMSO (final concentration 0.1%). Top: Confocal projection image of an individual neuron before (0’) and after (2’and 50’) the addition of PTEN-PDZ peptide, demonstrating initial accumulation of the peptide at the plasma membrane (2’), followed by diffusion into the cytosol (50’). Bottom: brightfield images of the same neuron. c. N-terminus biotinylated PTEN PDZ peptide was overlaid onto an array of PDZ domains. Bound peptide was visualized with the ABC kit from Vectasatin, based on its biotin modification. “Control” represents signal detected without peptide incubation. Uncropped version of the blot is shown in Supplementary Fig. 10. d. GST pull-down of recombinant PTEN bound to a GST fusion of the PDZ2 domain of hDlg, or to GST alone (left-most lane). GST beads were incubated with recombinant PTEN in the presence of 200 μM of the indicated peptide, or in the absence of peptide, as control. Pulled-down PTEN was then visualized by western blot, with an anti-PTEN antibody. Binding is normalized to the conditions without peptide incubation. Uncropped version of the western blot is shown in Supplementary Fig. 10. e. Top: superimposed representative fEPSPs evoked by stimulation of Schaffer Collaterals after 2 h incubation in Aβ42 assemblies (or vehicle control) with or without preincubation (0.5 h) with 10 μM scrambled peptide at 10, 50, 100, 150 and 200 μA of stimulation intensity. Bottom: Input-output curves of fEPSPs. P values were determined with two-way ANOVA with Bonferroni post-tests (results without peptide preincubation are replotted from Fig. 8e as lines). Scale bars: 0.5 mV, 20 ms. Data are represented as mean +/− SEM.