Fig. 7.

Translation initiation block is not responsible for inhibitory effect of ROS. a, b, d–f Incorporation of [³⁵S]-labelled amino acids in yeast or mammalian cells. Total cell extracts were separated by SDS-PAGE and analysed by autoradiography or immunodecorated with specific antibodies. a Wild-type (BY4741) yeast cells were grown on fermentative medium supplemented with 100 mM N-acetylcysteine (NAC) for 4 h. During the last 30 min of culturing, H₂O₂ was added. b HEK 293 cells were treated for 2 h with H₂O₂ in the presence of NAC as indicated. c Yeast cells were grown on respiratory medium at 19 °C and shifted for 2 or 3 h to restrictive temperature (37 °C). Samples were analysed by immunoblotting using specific antibodies. d HEK 293 cells were treated for 3 h with the PERK kinase inhibitor GSK2606414. H₂O₂ was added after 1 h of treatment with GSK2606414. e Wild-type (YPH499) yeast cells were treated for 3 h with 25 µM MG132. H₂O₂ was added 30 min prior harvesting of cells. f Wild-type (YPH499) yeast cells were treated with H₂O₂ for 30 min. Cells were washed and further incubated for 1 h. Incorporation of [³⁵S]-labelled amino acids was done at the same time as treatment with H₂O₂ or for 1 h starting after washing. WT, wild-type