FIGURE 4.

PP1 does not form a stable complex with CRMP2. A, SH-SY5Y cells were transfected with equivalent amounts of cDNA encoding green fluorescent protein (GFP), FLAG-CRMP2 (WT), or FLAG-CRMP2-S522A. After 16 h the cells were serum-starved for 8 h, then treated without or with 50 ng/ml IGF1 for 15 min. The CRMP2 proteins were immunoprecipitated (IP), then subjected to Western blot (IB) analysis using total CRMP2 (upper panel) or PP1 (lower panel) antibodies. Lys denotes total SH-SY5Y transfected lysate. B, non-transfected SH-SY5Y cell lysates treated without or with IGF1 were incubated with microcystin-Sepharose to pull down endogenous PP1, then subjected to Western blot analysis as in A.