Figure 2.

mTORC1 Fails to Localize to Lysosomes during Mitosis

(A) Asynchronous HeLa cells were treated with 1 μM AZD8055 for 2 h. Primary antibodies used were Lamp2 (magenta) and mTOR (green).

(B) HAP1 RAPTOR-GFP cells were treated with AZD8055 (1 μM), starved, or starved and then refed for 5 min. The primary antibody used was LAMP2 (magenta). Mitotic cells are indicated (M) in the DAPI channel.

(C) Asynchronous HeLa cells were immunostained for RagC (green) and Lamp2 (magenta).

(D) Asynchronous HeLa cells were transfected with either WT Rag duplex or active Rag duplex. Transfected cells are indicated with an X in the merge image (based on HA immunostain).

(E) HAP1 RAPTOR-GFP cells were treated with 50 nM paclitaxel (16 h), 1μM AZD8055 (2 h), or starved (2 h) prior to immunoprecipitation with GFP-TRAP. HAP1 parental cells (P) served as a negative control (P).

(F) HAP1 RAPTOR-GFP cells were treated with 50 nM paclitaxel (16 h), 2 μM RO-3306 (2 h), or starved (2 h) prior to immunoprecipitation with GFP-TRAP.

(G) HeLa cells stably expressing indicated HA-RAPTOR constructs were treated with 50 nM paclitaxel (16 h) prior to immunoprecipitation with HA antibody. Western blots are from a single experiment representative of three independent experiments.

Scale bars, 20 μm.