Fig. 5. Effect of GFP-MSK1 overexpression on substrate phosphorylation. (A) Quiescent wild-type (lanes 1-9) or GFP-MSK1-overexpressing (lanes 10-18) cells were stimulated with 50 ng/ml EGF (lanes 2, 3 and 11, 12), 25 ng/ml sAn (lanes 4-6, 13-15) or 100 nM TPA (lanes 7-9, 16-18), for the times indicated. Acid-extracted proteins were separated on 15% SDS-PAGE and Coomassie-stained (panel vi) or transferred to PVDF for western blotting. Blotting was carried out using anti-phosphoS10-H3 (panel i); anti-phosphoS10-H3occ (panel ii); anti-phosphoacetyl-H3 (panel iii); anti-phosphoS28-H3 (panel iv); or anti-phosphoHMG-14 (panel v). (B) Quiescent wild-type (lanes 1-4) or GFP-MSK1-overexpressing (lanes 5-8) cells were stimulated with EGF (50 ng/ml, 15 minutes; lanes 2, 6), sAn (25 ng/ml, 30 minutes; lanes 3, 7), or TPA (100 nM, 30 minutes; lanes 4, 8), or left untreated (lanes 1, 5). Acid-extracted proteins were separated on 15% SDS-PAGE (panel ii) or 20% acid-urea (panel i) gels and transferred to PVDF membranes for western blotting. Blotting was carried out with anti-HMG-14 antibody (panels i,ii). In i, the lower and upper arrows indicate the positions of the unphosphorylated and phosphorylated proteins, respectively. (C) Quiescent wild-type (lanes 1-4) or GFP-MSK1-overexpressing (lanes 5-8) cells were stimulated with EGF (50 ng/ml, 15 minutes; lanes 2, 6), sAn (25 ng/ml, 30 minutes; lanes 3, 7), or TPA (100 nM, 30 minutes; lanes 4, 8), or left untreated (lanes 1, 5). Cells were lysed in high-salt buffer (see Materials and Methods). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. Western blotting was carried out with an antibody which recognises CREB phosphorylated at S133 and ATF1 phosphorylated at S63 (panel i) or anti-ERK antibody (panel ii). In panel i, the arrows indicate the positions of phospho-CREB and phospho-ATF1.