Scrambled control sirna

S6K1 siRNA ON-TARGETplus SMARTpool was synthesized by Dharmacon and purchased from Fisher Scientific (Pittsburg, PA). This S6K1 siRNA pool containing three different siRNAs has been previously shown to efficiently suppress S6K1 expression^{62 (link), 63 (link)}. TAK1 and ATG7 siRNAs were purchased from Cell Signaling Technology (Danvers, MA). A scrambled control siRNA was purchased from Life Technologies (Invitrogen Life Technologies, Grand Island, NY). NSC34 cells seeded in 6-well plates were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen Life Technologies, Grand Island, NY) according to the manufacturer’s instruction. After incubation for 48 h, the cells were collected and analyzed for the expression of S6K1 and ATG7 and other relevant proteins by western blot. To determine the effect of ATG7 on A77 1726-induced SOD1 degradation, NSC34 cells were first transfected with control or ATG7 siRNA using Lipofectamine RNAiMAX, followed by transfection with SOD1-GFP or SOD1^G93A-GFP expression vector. After incubation for 24 h, the cells were left untreated or treated with A77 1726 for 24 h. Insoluble fractions of cell lysates were prepared and analyzed for SOD1 expression.

Wt MEFs were seeded into 12-well plates at a density of 0.75 × 10⁵ cells/well and after 18 h transfected with three different siRNA sequences targeting AMPKα1 (40 pmol each, #LQ-041035-00-0005, from Dharmacon^TM via THP Medical Products, Vienna, Austria) or scrambled control siRNA (from Invitrogen) using Lipofectamine RNAimax (Invitrogen) according to the manufacturers’ instructions. After 48 h, cells were lysed and immunoblotted for AMPK, Bach1 and actin.