Figure 7. NEDD4 knockdown toxicity is dependent on RTP801 protein.

DIV 5 primary rat cortical neurons were infected with lentiviruses containing a scrambled shRNA (ShCt) or a shRNA against RTP801 (ShRTP801). Two days later, neurons were transduced with a mix of three shRNA sequences against NEDD4 (ShNEDD4) or the corresponding control shRNA (ShCt). Cell lysates were analyzed 4 days later by Western Blot. Membranes were incubated with RTP801, NEDD4, P-AKT (S473), AKT, P-S6 (S235/236), ERK1/2 and α-spectrin antibodies, and with α-actin antibody as loading control. For α-spectrin the caspase-cleaved fragment (spectrin breakdown product 120, SBDP120) is shown. Representative immunoblots are shown along with densitometries (mean ± SEM) of at least two independent experiments done in triplicate. One-way ANOVA with Newman-Keuls multiple comparison test, *P < 0.05, **P < 0.01 versus ShCt/ShCt and ^#P < 0.05, ^##P < 0.01 versus ShCt/ShNEDD4.