Fig. 5.

S6 kinases promote the phosphorylation of eIF2α and translation inhibition. a Immunoblotting of HeLa cell lysates for Ser51 phosphorylation on eIF2α (p-eIF2α) after treating cells with or without 30 μM NaAsO₂ for 1 h in the presence of either DMSO or 50 nM rapamycin. Levels of phosphorylated RPS6 (p-RPS6) and S6 kinases (p-S6K) are shown to indicate the effectiveness of rapamycin at inhibiting mTORC1 activity. Blots for total eIF2α, RPS6, S6K1 and S6K2 protein levels are also presented. Quantification of p-eIF2α band intensities from 4 independent experiments is shown. The p-eIF2α levels were normalised against β-tubulin levels and are presented as relative increase in phosphorylation between untreated samples and those treated with NaAsO₂. b HeLa cells were treated with 30 μΜ NaAsO₂ for 30 min and the incorporation of puromycin into newly synthesised protein was assessed by immunoblotting. Band intensities for each lane were measured in 4 independent experiments and normalised against the intensity of Coomassie blue staining and presented as % translation inhibition. c HeLa cells transfected with siRNAs against S6K1 or S6K2 were treated with either 30 μΜ NaAsO₂ for 1 h or 0.5 mM NaAsO₂ for 30 min. Protein extracts were immunoblotted for p-eIF2α and total eIF2α protein levels. Quantification of p-eIF2α band intensities from 5 independent experiments is shown. The p-eIF2α levels were normalised against β-tubulin levels. d, e HeLa cells transfected with siRNAs against S6K1 or S6K2 were treated with either 30 μΜ NaAsO₂ for 1 h or 0.5 mM NaAsO₂ for 30 min and the incorporation of puromycin into newly synthesised protein was assessed by immunoblotting. Band intensities for each lane were measured in 3 independent experiments and normalised against the intensity of Coomassie blue staining and presented as % translation inhibition (e). f, g HeLa cells transfected with empty plasmids or plasmids expressing HA-S6K1p70 or HA-S6K2p54 were assessed for the incorporation of puromycin into newly synthesised protein by immunoblotting. The expression of the S6 kinases increased the levels of phosphorylated RPS6 (p-RPS6). Quantification of puromycin incorporation normalised against the intensity of Coomassie blue staining in 3 independent experiments is presented (g). Error bars are s.e.m and the data analysed using one-way Anova (*p < 0.04; **p < 0.0002)