Figure 5

Inhibition of ROCK2 increases Aβ40 and Aβ42 generation. A. N2A APPSweΔ9PS1 cells were grown to confluency and treated with vehicle or 100 nM RL-1 for 60 mins. After 60 mins equal volumes of media were collected and Aβ40 and Aβ42 levels were determined by sandwich ELISA (Wako). Aβ40 (p = 0.007) and Aβ42 (p = 0.03) levels were increased following treatment with 100 nM of the specific ROCK2 inhibitor, RL-1. Data was collected in duplicate or triplicate from 3 independent iterations (*p > 0.05 **p < 0.01). B. N2A APP SweΔ9PS1 cells were transduced with non-specific scrambled shRNA or 4 different shRNA ROCK2. 72 hrs post transduction media and cell lysates were collected and snap frozen. Equal volume and concentrations of media and lysate respectively were analyzed by SDS PAGE and transferred to PVDF. Lysates were analyzed for ROCK2 and GFP as a control for transduction efficiency and media analyzed for sAPPα and Aβ. Protein levels are expressed as percentage of non-transduced C (C). Knockdown of ROCK2 resulted in increased generation of Aβ and sAPPα with all shRNA ROCK2 viruses analyzed but not after transduction with the scrambled non-specific control shRNA.