Fig. 3

InRapa recovers mTOR hyperactivation and induce autophagy. (a 1–8) Representative immunofluorescent images showing p-mTOR(Ser2448) signal in the CA3 region of the hippocampus from euploid mice treated with Veh and InRapa (A1–4), and Ts65Dn mice treated with Veh and InRapa (A5–8). DAPI (blue) was used to identify cell nuclei. Scale bar represent 20 μm. (b) Quantification of fluorescence signal. (c) Representative WB showing hippocampal p-mTOR (Ser 2448,2481) and mTOR total protein levels, p-P70S6K and P70S6K total protein levels, Atg5, Atg7, Beclin, LC3II protein levels. (d) Quantification of panel C showing mTOR protein levels, p-mTOR (Ser2448)/mTOR ratio and p mTOR (Ser2481)/mTOR ratio. (e) Quantification of panel C showing P70S6K and p-P70S6K/ P70S6K ratio. (f) Quantification of panel C showing LC3II and Beclin protein levels. (g) Quantification of panel C showing Atg5, Atg7 protein levels and quantification of the complex Atg5-Atg12. (h-j) Quantification of mRNA levels of mTOR (h), LC3 (i) and Beclin (j) analyzed by RT-PCR analysis. Densitometry values shown in the bar graph are the mean of 8 (WB) and 6 (RT-PCR) samples per each group normalized per total load and are given as percentage of Eu Veh, set as 100%. (*p < 0.05, **p < 0.01, ***p < 0.001)