FIG. 3.

CaM antagonists have a transient inhibitory effect on the phosphorylation of ERKs induced by NGF. (A) PC12 cells were pretreated (+) or not pretreated (−) for 1 h with W13 and then stimulated (+) or not stimulated (−) with NGF for the indicated times. After treatment, cells were lysed and protein extracts were analyzed as described in the legend to Fig. 1 to detect ERK phosphorylation. (B) PC12 cells were treated as described in panel A for the indicated times, and protein extracts were analyzed by Western blotting with an anti-phospho-RSK antibody (P-RSK) and an anti-phospho-CREB antibody (P-CREB) and stripped and reprobed with an anti-CREB antibody (CREB) as a control for the protein content per lane. (C) PC12 cells were pretreated (+) or not pretreated (−) for 1 h with W12 or W13 and then stimulated (+) or not stimulated (−) for 1 h with NGF. After treatment, cells were lysed and protein extracts were analyzed by Western blotting with an anti-c-Fos antibody (upper panel) and stripped and reprobed with an anti-α-tubulin antibody (lower panel) as a control for the protein content per lane. ∗, positive control for CREB phosphorylation obtained from the antibody supplier (total cell extracts from SK-N-MC cells treated with Forskolin).