The Akt-mTOR pathway is more activated in CLN3 and especially in CLN2 fibroblasts.

(A) Control (CTRL), CLN2 and CLN3 fibroblasts were incubated in high (H) and low (L) proteolysis media. Lysates were analyzed by western blot using antibodies against the phosphorylated forms of various proteins upstream and downstream of mTOR: Akt-Thr 308 (P-Akt-T), Akt-Ser 473 (P-Akt-S), p-38α (P-p38α), ERK 1/2 (P-ERK 1/2), p70S6K (P-p70S6K) and 4EBP1 (P-4EBP1) and their respective total proteins. The bands corresponding to the phosphorylated proteins, obtained from three different experiments, were densitometred and normalized to the corresponding bands of total protein. Data are plotted in percentage relative to the highest value. Stars indicate statistically significant differences from control values under high and low proteolysis conditions (*p<0.05, **p<0.01 and ***p<0.005). (B) Control, CLN2 and CLN3 fibroblasts were incubated for the indicated times in high proteolysis medium with EGF (100 ng/ml). Total extracts were analyzed using P-Akt-T and total Akt antibodies. A representative blot is shown. The bands corresponding to phosphorylated and total Akt were quantified by densitometry as in A). Stars indicate statistically significant differences from control values at each time (***p<0.005).