FIG 5.

Ulk1 is phosphorylated at S757, while mTOR kinase activity is partially suppressed under hyperosmotic stress. (A) Wild-type MEFs and T24 cells were cultured with amino acid-free DMEM (−AA) or Hanks balanced salt solution (HBSS) or DMEM with 0.8 M sucrose, 0.8 M mannitol, 0.4 M NaCl, or 0.4 M KCl for 1 h. A sample without the cultures was also included in the leftmost lane (0 h). Cells were lysed for Western blot analyses using antibodies against phosphorylated mTOR (S2448), mTOR, phosphorylated Ulk1 (S575), Ulk1, phosphorylated S6K1 (T389), S6K1, phosphorylated 4E-BP1 (T37/46), 4E-BP1, and GAPDH as a loading control. Molecular weights are indicated on the right. (B) The experiments were repeated 3 times. Band intensities were quantified, and the ratio of the phosphorylated form to total protein was calculated, which was then compared to the control value. Statistical significance between the control and each condition was determined by Student's t test. *, P < 0.05; **, P < 0.01; NS, not significant.