Figure 5
From: TAK1 regulates autophagic cell death by suppressing the phosphorylation of p70 S6 kinase 1
TAK1 binds to S6K1 and suppresses the phosphorylation of S6K1.
(A) Phosphorylation of dS6K was detected by immunoblotting using a p-dS6K antibody in third-instar larvae and pupae of the indicated genotypes. Protein extracts from pupae and third-instar larvae were used. β-tubulin was used as a loading control and Rheb-overexpressing flies were used as positive controls. dS6K phosphorylation in each genotype was quantified using Adobe Photoshop and normalized to β-tubulin. (B) Cells were transfected with the indicated constructs and incubated for 48 h. Cell lysates were then collected and immunoblotted with the indicated antibodies. S6K1 phosphorylation decreased in a TAK1 dose-dependent manner. (C) HEK 293T cells were transfected with the indicated constructs and 48 h after transfection, a Myc immunoprecipitate was obtained and analyzed for the presence of an S6K1-TAK1 complex using a Flag antibody. The blots in the lower four panels were derived from the same cell lysates using the indicated antibodies. (D) Cells were transfected with the denoted constructs and 48 h after transfection, S6K1-TAK1 complex was detected using an S6K1 antibody. The blots in the lower three panels were derived from the same cell lysates using the indicated antibodies. (E) Myc-S6K1 construct was co-transfected into cells with Flag-tagged TAK1-WT or TAK1-KW and incubated for 48 h. Lysates were collected and immunoblotted with the indicated antibodies.
