Extended Data Fig. 4: mTORC1 phosphorylates Cdh1.

a, MCF-10A cells were starved for 48 h to induce quiescence followed by mitogen stimulation for 4 h. Cells were then collected and lysed. Whole cell extracts were immunoprecipitated with an mTOR antibody and probed for the indicated proteins. Representative blot from n = 2 independent experiments. b, MCF-10A cells were starved for 48 h to induce quiescence followed by mitogen stimulation for 4 h. Cells were then collected and lysed. Whole cell extracts were immunoprecipitated with either Cdh1 or EGFR antibody and probed for the indicated proteins. Representative blot from n = 2 independent experiments. c, MCF-10A cells were starved for 48 h to induce quiescence followed by mitogen stimulation for 4 h. Cells were then collected and lysed. Whole cell extracts were immunoprecipitated with HA as a negative control or with antibodies to either Cdh1 (top) or mTOR (middle) and probed for the indicated proteins. Whole cell lysates are shown at the bottom. Representative blot from n = 2 independent experiments. d, Coomassie stained SDS-PAGE gels of 400 ng of the indicated purified proteins. e, In vitro kinase assay with purified His-Sumo-Cdh1 from bacteria and recombinant mTOR-mLST8. Kinase reactions were resolved on SDS-PAGE and probed with a pan phospho Serine/Threonine/Tyrosine antibody. Representative blot from n = 3 independent experiments. f, In vitro kinase assay of untagged Cdh1-WT, Cdh1-T129A, and Cdh1-T129D with recombinant GST-ERK. Kinase reactions were resolved on SDS-PAGE and probed with a pan phospho-threonine antibody. Representative blot from n = 2 independent experiments. g, In vitro kinase assay with untagged Cdh1-WT, Cdh1-T129A, and Cdh1-T129D with recombinant GST-Cdk1-Cyclin B. Kinase reactions were resolved on SDS-PAGE and probed with a pan phospho-threonine antibody. Representative blot from n = 2 independent experiments. h, In vitro kinase assay with untagged H1 and recombinant GST-Cdk1-cyclin B. Kinase reactions were resolved on SDS-PAGE and probed with a pan phospho-serine/threonine/tyrosine antibody. Representative blot from n = 2 independent experiments. i, In vitro kinase assay with untagged Cdh1-WT, Cdh1-T129A, and Cdh1-T129D with recombinant FLAG-DYRK4. Kinase reactions were resolved on SDS-PAGE and probed with a pan phospho-threonine antibody. Representative blot from n = 2 independent experiments. j, Cells were starved for 48 h to induce quiescence, followed by mitogen stimulation with or without rapamycin. Whole cell lysates were immunoprecipitated with anti-Cdh1 antibody and probed for indicated proteins. Representative blot from n = 2 independent experiments.