Journal: Neurobiology of disease

Article Title: A peptide inhibitor of Tau-SH3 interactions ameliorates amyloid-β toxicity

doi: 10.1016/j.nbd.2019.104668

Figure Lengend Snippet: Tau-PxxP 5/6 inhibits the Tau-Fyn interaction in cells. A: Timeline of Tau-Fyn PLA experiments in HEK-293 cells. B: Representative immunofluorescent images of HEK-293 cells treated with Tau-PxxP 5/6 (rrrqrrkkrgRSRTPSLPTPPT-REPKK) or Tau-CTD (rrrqrrkkrgENAKAKTDHGAEIVYKS) after Tau-Fyn proximity ligation assay (PLA) was performed (full description in the section). Green puncta represent sites of Tau-Fyn interaction, red is Tau-mKate2, and magenta is immunofluorescently labeled Fyn. Scale bar = 10 μm. Quantification of Tau-Fyn PLA density demonstrates that Tau-SH3 inhibitor Tau-PxxP 5/6 reduced Tau-Fyn interaction by 40% while negative control peptide Tau-CTD did not (ANOVA, F(2, 33) = 10.14, p = .0004, average of 5–7 images per coverslip from n =11–13 coverslips from N = 6 distinct passages, see for details of quantification and analysis). Differences indicated: ** p < .01, and *** p < .001 by Dunnetťs post hoc test with correction for multiple comparisons relative to control. C: Western blot of lysate from HEK-293 cells demonstrates that Tau-PxxP 5/6 did not change Tau, Fyn, or GAPDH levels (Studenťs t -test, t(10)=1.86, p = .092; t(9)=0.28, p = .79; t(10) =1.78, p = .10, respectively, n =5–6 wells per group).

Article Snippet: Trypsin was inactivated by diluting it 1:10 in DMEM (Fisher MT10013CV) supplemented with 10% premium select fetal bovine serum and 1% Pen Strep (ThermoFisher 15140122) and the cells were plated to 10% confluency in 500 μL supplemented DMEM onto 12 mm No 1 glass coverslips (Carolina Biological 633029) coated overnight at 4 °C with 0.1 mg/mL Poly-D-Lysine and 0.02 mg/mL laminin in a 24-well plate (Fisher 08–772–1).

Techniques: Proximity Ligation Assay, Labeling, Negative Control, Control, Western Blot

Journal: Neurobiology of disease

Article Title: A peptide inhibitor of Tau-SH3 interactions ameliorates amyloid-β toxicity

doi: 10.1016/j.nbd.2019.104668

Figure Lengend Snippet: Tau-PxxP 5/6 inhibits endogenous Tau-Fyn interaction in neurons. A: Representative immunofluorescent image of DIV21 primary neurons after Tau-Fyn PLA. Cyan puncta represent sites of Tau-Fyn interaction and red is immunofluorescently labeled Tau. Scale bar = 50 μm. Inset: magnification of a neurite showing Tau-Fyn PLA (cyan) adjacent to Tau-positive neurites (red). Scale bar = 5 μm. B: Timeline of endogenous Tau-Fyn PLA inhibition experiments. C: Representative images of endogenous Tau-Fyn PLA after Tau-PxxP 5/6 treatment demonstrating reduction of endogenous Tau-Fyn PLA in neurons by Tau-PxxP 5/6 . Tau-PxxP 5/6 reduced endogenous Tau-Fyn interaction in neurons by 62% (Studenťs t -test, t(13) = 6.124, p < .0001, average of 5–7 images per coverslip from n =7–8 coverslips). Scale bar = 25 μm.

Article Snippet: Trypsin was inactivated by diluting it 1:10 in DMEM (Fisher MT10013CV) supplemented with 10% premium select fetal bovine serum and 1% Pen Strep (ThermoFisher 15140122) and the cells were plated to 10% confluency in 500 μL supplemented DMEM onto 12 mm No 1 glass coverslips (Carolina Biological 633029) coated overnight at 4 °C with 0.1 mg/mL Poly-D-Lysine and 0.02 mg/mL laminin in a 24-well plate (Fisher 08–772–1).

Techniques: Labeling, Inhibition