Figure 4.

MEF2A-CSox can selectively report on p38α activation in unfractionated cell lysates. Dose dependant responses to the addition of SB203580 (a) and BIRB796 (b) were observed in sorbitol-stimulated lysates (10 µg) using MEF2A-CSox (1 µM). The observed IC₅₀ and structure of each compound are shown in the inset. c) Phosphorylation reactions were performed on lysates which had been depleted using a p38α-specific antibody. Values were background subtracted using the activity remaining in the input lysate after addition of 1 µM SB203580. The inset shows a western blot of the immunodepleted lysates. d) The indicated cell lines were stimulated with 300 mM sorbitol for 1 hr (S). Phosphorylation reactions were performed as above and compared to lysates from cells which had not been stimulated (C). The amount of p38α activity in each individual lysate was determined by subtracting the activity remaining after the addition of 1 µM SB203580. Western blots for both total and active p38 are shown above each sample. All reactions contained 1 µM staurosporine.