Figure 6. Effect of N-acetyl-L-cysteine on the activity of ERK8 and its phosphorylation at the Thr-Glu-Tyr motif in response to hydrogen peroxide or osmotic shock in HEK293 cells.

(A) The cells were transfected with a vector encoding wild-type HA–ERK8 as in Figure 4(A) and at 36 h post-transfection incubated for 15 min with (grey bars) or without (black bars) 10 mM N-acetyl-L-cysteine (NAC). The cell media were aspirated, the cells were washed at 21 °C with PBS and fresh media was added. Cells were then exposed for 10 min to either 1 mM hydrogen peroxide or 0.5 M sorbitol and lysed. ERK8 was immunoprecipitated from cell extracts using an anti-HA antibody and its activity was measured using MBP as substrate. (B and C) The cell lysates from (A) were analysed by immunoblotting, as in Figure 4(A). (D) The cells were first transfected as in (A). At 36 h post-transfection they were then incubated for 10 min without (lanes A and B) or with (lanes C–H) 1 mM hydrogen peroxide. Either the cells were lysed (lanes A–D) or the culture medium was aspirated and the cells washed at 21 °C with PBS and fresh media was added (lanes E–H). The cells were then incubated for 30 min without (lanes E and F) or with (lanes G and H) 10 mM N-acetyl-L-cysteine.