Supplementary Figure 4: siRNA-directed knockdown of Lamp2a reduces TAT-GluN2BCTM induced DAPK1 degradation.
From: Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation
(a) DNA electrophoresis of Lamp2a showing siRNA (60pmol)-directed knockdown of Lamp2a 3d after siRNA treatment. Scrb-siRNA: scrambled siRNA, LAMP-2A-siRNA: Lamp2a-targeting siRNA. N=3. (b-c) Sequential immunoblotting showing the inhibition of TAT-GluN2BCTM induced knockdown of DAPK1 by specifically blocking CMA with siRNA-mediated Lamp2a knockdown. 3d following siRNA treatment, cells were incubated in TAT-GluN2BCTM (25μM) for 1hr before and during NMDA (50μM; 30min). Cells were harvested 2h after the peptide and NMDA washout. β-actin was used as loading control. Lamp2aknockdown: n=7, One-way ANOVA p<0.001, F(3,24)=13.455; DAPK1 Knockdown: n=7, Kruskal-Wallis One-Way ANOVA on Ranks with Tukey post hoc, p=0.002, H(3)=15.047; * compared to saline control, Δ compared to NMDA-treated group (grey bar). *,Δ p<0.05, ***p<0.001; bars represent relative mean values±s.e.m. normalized to saline control (arbitrarily set as 1).Cells were collected from at least 3 separate primary cultures. Full-length blots are available in Supplementary Figure 9.
