Figure 5.

Inactivation of autophagic pathway by constitutively active Rheb in NIH 3T3 cells grown under tyrosine deficiency. (A) NIH 3T3 cells (1 × 10⁶) grown in complete media were transiently cotransfected with Flag-RhebQ64L (0.8 µg), GFP-LC3 (0.1 µg) and HA-S6K (0.1 µg). At 16 h after transfection, culture media were replaced with fresh DMEM or tyrosine-deficient media (-Tyr) and incubated for additional 24 h. The phosphorylation of ectopically expressed S6K (HA-S6K) and conversion of GFP-LC3-I into GFP-LC3-II were examined by protein gel blotting. (B) Band intensities were quantified and the ratio of p-S6K/S6K or LC3-II/LC3-I was shown. (n = 3 experiments). Values represent means ± SD, *p < 0.05.