Extended Data Figure 2. Dimerization deficient CASTOR1 mutants bind arginine but fail to inhibit mTORC1 in cells.
a) The dimerization deficient CASTOR1 Y207S and I202E mutants bind arginine in vitro. FLAG-immunoprecipitates prepared from HEK-293T cells transiently expressing indicated FLAG-tagged proteins were used in binding assays with [3H]Arginine as described in the methods. Unlabeled arginine was included as a competitor where indicated. Values are Mean ± SD for 3 technical replicates from one representative experiment.
b) Dimerization deficient CASTOR1 Y207S and I202E mutants fail to inhibit mTORC1. HEK-293T cells transiently expressing FLAG-S6K1 and HA-tagged CASTOR1 WT, Y207S, or I202E were starved of arginine for 50 min and, where indicated, restimulated for 10 min. FLAG- immunoprecipitates were prepared from lysates and analyzed as in 1C. Phospho-S6K1 was used as an indicator of mTORC1 activity.