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. Author manuscript; available in PMC: 2016 Apr 21.
Published in final edited form as: Cell Rep. 2015 Apr 9;11(3):446–459. doi: 10.1016/j.celrep.2015.03.040

Figure 1. Developing a yeast system to assay for mTOR kinase inhibition.

Figure 1

(A) Wild type (WT) yeast cells were spread onto YPD plates and tested for sensitivity to structurally diverse mTOR kinase inhibitors by disc halo assay. Rapamycin was used as a positive control.

(B) The N-terminus of TOR2 (1-2080 aa) was fused in frame with mTOR kinase domain (2140-2549 aa). The TOR2-mTOR fusion is expressed under the control of TOR2 promoter in a centromeric plasmid.

(C) Yeast strain expressing WT TOR2 or TOR2-mTOR fusion was analyzed for expression by immunoblot with an antibody specific for mTOR kinase domain. PGK1 was used as a loading control and extracts from MCF7 breast cells were used as a positive control for mTOR.

(D) TOR2-mTOR fusion was expressed in tor2-dg and tested for its ability to complement TOR2 function by growth at permissive and restrictive temperatures.

(E) tor2-dg cells expressing TOR2 or TOR2-mTOR were serially diluted by 10-fold and tested for drug sensitivity on plates containing BEZ235 and OSI-027.