Fig 4.
UCH-L1 disrupts the DDB1-raptor complex that is required for mTORC1 stability. (A to C) 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. (D) KMS-28doxsh cells were incubated with or without doxycycline for 5 days to deplete UCH-L1 and were then subjected to immunoprecipitation with the indicated antibodies. The precipitates were then immunoblotted using the indicated antibodies. (E and F) mTOR complexes were immunoprecipitated from 293T cells, washed extensively, and incubated with purified UCH-L1. Where indicated, NEM was included to inactivate catalytic activity. After extensive washing, the remaining bound proteins were analyzed by immunoblotting using the indicated antibodies.