The mTOR functional analysis and purification of a Tsc1/Tsc2 complex. (a) Autoradiograph from an mTOR kinase reaction. Addition of mTOR and Tsc1/Tsc2 are indicated at the top. Equivalent amounts of mTOR autokinase activity and kinase activity on 4E-BP1 are seen whenever mTOR is included. C, anti-C20 tuberin Ab; N, anti-N19 tuberin Ab; E, eluate. (b) Autoradiograph of a phosphatase assay. 4E-BP1 was phosphorylated in vitro using γ³²P-ATP and then was included as a substrate to assess phosphatase activity of two TP53^–/–Tsc2^–/– and two TP53^–/– control cell line extracts. There is no difference in the level of phosphatase activity. (c) Coomassie blue–stained gel showing successive steps in the purification of TSC1/TSC2 from brain extracts. Material bound to an anti-TSC1 affinity (H2 Ab) column, residual on the column after elution with peptide, the eluate, and the material obtained from an anti-TSC2 (C20) Ab column are shown in successive lanes. The location of 14-3-3γ is indicated by an asterisk. (d) Immunoblot analysis of Tsc1/Tsc2–binding partners. IP was performed with the indicated Ab’s (Tsc2 N19, C20) followed by immunoblotting. Tsc2 row: + indicates extract from a control TP53^–/– cell line; – indicates a Tsc2^–/–TP53^–/– cell line.