Overexpression of RagB^GTP can derepress TOP mRNA translation in amino acid-starved cells, but not in cells deprived of oxygen.

(A) HEK293 cells were infected with lentiviral expression vectors encoding FLAG-RagB or FLAG-RagB^GTP. 48 h post infection cells were subjected to selection by puromycin and 48 h later were either kept untreated (+), amino acid-starved for 8 h (−AA), amino acid starved during the last 3 h of 24 h serum starvation (–Ser/AA) or deprived of oxygen (−O₂) for 16 h. Cells were harvested and their cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (B) Polysomal analysis of cytoplasmic extracts from cells treated as describe in (A).