Fig 3.
UCH-L1 reorganizes the assembly of competing mTOR complexes. (A) KMS-28doxsh cells were incubated with or without doxycycline for 5 days to deplete UCH-L1 and were then subjected to immunoprecipitation (IP) with the indicated antibodies. Ratios were determined using ImageJ. (B) Brain tissue was harvested from age-matched mice of the indicated genotypes and subjected to immunoprecipitation using the indicated antibodies. The precipitates were then immunoblotted using the indicated antibodies. Ratios were determined using ImageJ. (C and D) HEK-293T cells, stably transduced with the indicated constructs, were subjected to immunoprecipitation using the indicated antibodies. The precipitates were then immunoblotted using the indicated antibodies. (E) mTORC1 was purified from HEK-293T cells by immunoprecipitation using raptor or control IgG antibodies as indicated. Purified S6K was added to the resulting precipitates, and after further incubation, the reactions were stopped by adding 2× SDS sample buffer. The reaction mixtures were subjected to immunoblotting with the indicated antibodies. Where indicated, ([2×]), the amount of mTORC1 was increased 2-fold to equalize the amount of mTOR in the reaction mixture. (F) mTOR complexes were immunoprecipitated from 293T cells, washed extensively, and incubated with purified UCH-L1. Where indicated, N-ethylmaleimide (NEM) was included to inactivate catalytic activity. After extensive washing, the remaining bound proteins were analyzed by immunoblotting using the indicated antibodies.