Fig. 4.

Identification of putative MAPK substrates from brain PSD and neuron culture by 5557 immunoprecipitation. A, proteins immunoprecipitated (IP) by antibody 5557 from cortical culture (P2 fraction solubilized in RIPA buffer) or rat brain (PSD1 fraction solubilized by boiling in 1% SDS) were visualized and quantified by silver staining. Okadaic acid treatment of cortical culture (0.1 μm, 1 h) changed the pattern immunopurified by 5557 (lane 2). B, distribution of the abundance ratio log₂(OA/untreated) of proteins identified in OA-treated versus untreated cortical culture as an index for OA-induced protein phosphorylation. For all characteristic peptides of a protein the intensity ratios from MS survey scans were transformed into logarithmic (log₂) values and averaged. All proteins were binned into 0.5-unit intervals. The distribution was fitted with two summed Gaussian distributions (dashed lines; parameters as indicated: μ_1/2, mean, σ_1/2, S.D.). C, the percentage of proteins containing potential MAPK motifs (PX(S/T)P) increases with OA-induced enrichment. For comparison the dotted line denotes the basal frequency of 30% within the rat proteome (10,403 of 35,015; NCBI rat reference sequences, May 2007).