Fig. 4. PRAS40 knockdown decreases mTOR kinase activity towards S6K1.

For in vitro kinase activity, cells were lysed in 0.3% CHAPS buffer. mTOR was immunoprecipitated from 150 μg of cell lysate, and the activity was assayed using S6K1 as the substrate. Reaction mixtures were incubated as described under “Materials and Methods Section” in the presence (panel A) or absence (panel B) of [γ- ³²P] ATP. Panel B, Reaction mixtures were examined by Western blots using the anti-phosphorylated S6K1 (T389) antibody. Results were corrected with immunoprecipitated mTOR. Panel C, equal amounts of cell extracts were analyzed via Western blotting using antibodies that recognize either phosphorylated or total S6K1 and S6 ribosomal protein. Results are expressed as a percentage of scrambled control levels. Each bar graph represents mean ± SE of 4 independent experiments consisting of 4 replicate samples per experiment. * P< 0.05 versus the scrambled control values.