Fig. 2. GSK-3ß phosphorylates MAP1B on Ser1260. The serine residues at positions 1247, 1251, 1255, 1257 and 1260 (mouse sequence) were replaced by valine residues to produce recombinant proteins SP1 to SP5, respectively. Kinase assays were performed using the recombinant proteins as substrates. Recombinant proteins were incubated with a high-speed supernatant from neonatal rat brain (S1) in kinase buffer to investigate if they could be phosphorylated by GSK-3ß at the site recognised by the mAb SMI-31. Western immunoblot analysis of the kinase assay GST pull-downs showed that recombinants SP1 to SP4 were positive for the mAb SMI-31 epitope. However, the expression of the mAb SMI-31 epitope by the SP5 recombinant incubated with S1 was greatly reduced in comparison to the other recombinants, including SP (c.f. Fig. 1). This indicates that the serine at position 1260 in the full-length mouse MAP1B sequence is phosphorylated by GSK-3ß to produce the mAb SMI-31 epitope. Controls (Con) were treated in the same way but without the inclusion of S1. The same blot was then stripped and reprobed with an anti-GST antibody (GST) to assess the protein loading levels, which were found to be similar (lower panel).