Tak1 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 [57 (link), 58 (link)]. TAK1 siRNA was purchased from Cell Signaling Technology (Danvers, MA). A scrambled control siRNA was purchased from Life Technologies (Invitrogen Life Technologies, Grand Island, NY). A375 cells seeded in a 6-well plate were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen Life Technologies, Grand Island, NY) according to the manufacturer’s instruction. After incubation for 48 hr, the cells were harvested and analyzed for S6K1 expression and for the phosphorylation of S6K1, AKT, S6, AMPK, and ULK1 by Western blot.

TAK1 siRNA was purchased from Cell Signaling Technology (Danvers, MA). A scrambled control siRNA was purchased from Life Technologies (Invitrogen Life Technologies, Grand Island, NY). HeLa cells seeded in a six-well plate were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen Life Technologies) according to the manufacturer’s instruction. After incubation for 48 h, the cells were left uninfected or infected with 10 MOI and then incubated for 2 h. The cell lysates were prepared and analyzed for the expression of TAK1 and other relevant proteins.