The mTORC1 signaling pathway is not involved in SAM checkpoint activation. (A) p190 cells were shifted to -Met, -Leu, control or Cyc media. Cells were harvested at indicated time and analyzed by immunoblotting using antibodies directed against phospho-p70S6K (P-p70), phospho-S6 (P-S6) and tubulin. (B), p190 cells were shifted to -Met, -Leu or control media, or medium with 20 nM rapamycin (Ra) and analyzed as for A, and also for phospho-Akt Ser473 (P-Akt). (C) p190 cells were infected with a retrovirus carrying vehicle (p190Vehicle) or a Flag–RagBQ99L (p190RagBQ99L) expression cassette and selected with puromycin for stable expression. Selected cells were shifted to -Leu or rapamycin-containing media for the time intervals indicated and analyzed by immunoblotting to detect phospho-S6 and Flag–RagBQ99L protein levels. (D) p190Vehicle and p190RagBQ99L cells were shifted to -Met, control, and Cyc media for 12 hours and cell cycle profiles were analyzed by flow cytometry.