Fig. 3. The effects of double point mutations on GSK-3ß phosphorylation of MAP1B. To produce double point mutated recombinant proteins SP1/5, SP2/5, SPS/5, SP3/5, SP4/5 and SPT/5, the serine residues at positions 1247, 1251, 1253, 1255 and 1257 and Thr1265, respectively, were replaced by valine residues in the SP recombinant in which Ser1260 was replaced by a valine (see Table 1). 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. Recombinant proteins SP1/5, SP2/5, SP3/5 and SPS/5 were immunoreactive with mAb SMI-31 after incubation in the kinase assay as was SP5 itself. However, recombinant proteins SP4/5 and SPT/5 were not immunoreactive, or showed greatly reduced immunoreactivity, with mAb SMI-31. To enhance the relatively weak mAb SMI-31 signal of the SP5 recombinant protein the blots were overexposed, as can be seen from the SP control (c.f. Fig. 2). The same blot was then stripped and reprobed with an anti-GST antibody (GST) to assess the protein loading levels, which were similar (lower panel).