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. 2006 Aug 7;149(2):215–225. doi: 10.1038/sj.bjp.0706842

Figure 6.

Figure 6

Phosphorylation of ERK 1/2 and downstream MSK1 and CREB in ATRA-treated cells: role of ERK1/2 in the ATRA-induced increase of COX-2 protein expression. (a) Effect of preincubation with the MKK1 inhibitor PD098059 (50 μM/1 h) on the upregulation of COX-2 by ATRA (10 μM/24 h). Equal protein loading was confirmed by probing with an anti-α-actin antibody. Normalized density ratio of COX-2 over α-actin is indicated for each band. (b, c) Time course of the phosphorylation of ERK1/2 (P-ERK1/2) (B) and CREB (P-CREB) (c) following incubation with 10 μM ATRA. Equal protein loading was confirmed by probing with anti-total ERK2 or anti-total CREB antibodies, respectively. Normalized density ratio of ERK1/2 over total ERK2 or of P-CREB over total CREB is indicated for each band. (d) Effect of pre-incubation with the MKK1 inhibitor PD098059 (50 μM/1 h) on the phosphorylation of CREB (P-CREB) by ATRA 10 μM/24 h). Equal protein loading was confirmed by probing with anti-total CREB antibody. Normalized density ratio of P-CREB over total CREB is indicated for each band. (e) Phosphorylation of MSK1 (P-MSK1) following incubation with 10 μM ATRA. Equal protein loading was confirmed by probing with an anti-α-actin antibody. Normalized density ratio of P-MSK1 over α-actin is indicated for each band. (f) Effect of preincubation with the MSK-1 inhibitor H89 or the ERK1/2-activated ribosomal proteinS6 kinase-2 (RSK2) inhibitor GF109203X (10 μM each/1 h) on the upregulation of COX-2 by ATRA (10 μM/24 h). Equal protein loading was confirmed by probing with an anti-α-actin antibody. Normalized density ratio of COX-2 over α-actin is indicated for each band. Every photograph represents at least three repeated experiments.