Supplementary Figure 5: Pep1-mediated intracellular delivery of short synthetic GluN2B-CTM peptides specifically knocks down active native DAPK1 in cultured neurons.

(a) Schematic illustration of synthetic peptide GluN2B-CTM. (b) GluN2B-CTM, when delivered into cortical neurons, specifically decreases the level of native DAPK1 in an NMDA-stimulation dependent manner. GluN2B-CTM was first mixed with intracellular delivering carrier peptide Pep-1 at a 1:4 ratio for 30min to form a plasma membrane permeable peptide complex, which was then bath applied to neurons 60min prior to and during NMDA treatments (50μM; 30min). GluN2B-CTM plus Pep-1 (but not the Pep-1 alone) dose-dependently (b, p<0.001 F(7,22)=6.993, from at least 2 individual experiments) and time-dependently (c, p<0.001, F(7,28)=10.034, from at least 3 individual experiments) decreased the level of endogenous DAPK1 in cultured cortical neurons following NMDA treatment. The reduction required NMDA stimulation (b) and was rescued by inhibiting lysosome function with NH₄Cl (d; NH₄Cl 20mM; n=5; One-way ANOVA, p<0.001, F(3,16)=15.129.). Bars represent relative DAPK1 levels normalized to saline group (white bar, arbitrarily set as 1), and compared to both saline (white bar, *) or NMDA-treated group (grey bar, Δ). Membrane re-probing for β-actin was used as loading control. *^,Δ p<0.05, **^,ΔΔ p<0.01 and ***^{, ΔΔΔ}p<0.001; bars represent relative mean values±s.e.m. normalized to the saline control (white bar, arbitrarily set as 1). Cells were collected from at least 3 separate primary cultures. Full-length blots are available in Supplementary Figure 9.