FIG. 7.

CaM inhibitor W13 prevents ERK activation upstream of MEK. (A) PC12 cells were pretreated (+) or not pretreated (−) for 1 h with W13 and then stimulated (+) or not stimulated (−) for 5 and 45 min with NGF. After treatment, cells were lysed and protein extracts were analyzed by Western blotting with an anti-phospho-MEK1/2 antibody (upper panel) and stripped and reprobed with an anti-MEK1/2 antibody (lower panel) as a control for the protein content per lane. (B) PC12 cells were transfected with a constitutive form of MEK1 (CA-MEK) or with a plasmid control (MOCK) (see Materials and Methods). After 48 h of transfection, cells were treated (+) or not treated (−) with W13. After treatment, protein extracts were analyzed by Western blotting for ERK phosphorylation as described in the legend to Fig. 1. (C) PC12 cells were pretreated (+) or not pretreated (−) for 1 h with 25 μM PD098059 or with 0.1% Me₂SO as a vehicle and then were stimulated (+) or not stimulated (−) for 5 min with NGF. After treatment, cells were lysed and protein extracts were analyzed by Western blotting for ERK phosphorylation as described in the legend to Fig. 1.