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Fig. 4. Polyclonal antibodies BUGS and SuperBUGS recognise phosphorylated Ser1260 and phosphorylated Thr1265 in MAP1B respectively. Western immunoblot analysis of SP, SPT, SP5 and SPT/5 recombinant proteins that were incubated with a high-speed supernatant from neonatal rat brain (S1) in kinase buffer to investigate if they could be phosphorylated at the sites recognised by mAb SMI-31, pAb BUGS and pAb SuperBUGS. All three antibodies recognised native MAP1B in the S1 supernatant (arrowheads) and the SP recombinant protein following phosphorylation in the kinase assay. The recombinant protein SPT was not recognised by mAb SMI-31 or by pAb SuperBUGS but this mutation did not affect the binding of pAb BUGS, suggesting that the S1265V mutation did not affect GSK-3ß phosphorylation of Ser1260. In confirmation of earlier results, the SP5 recombinant showed a small, residual binding of mAb SMI-31. As expected, pAb BUGS did not recognise the SP5 recombinant protein whereas pAb SuperBUGS did. The double point-mutated recombinant protein SPT/5 was not recognised by any of the three antibodies. The same blot was stripped and reprobed with an anti-GST antibody (GST) to assess the protein loading levels, which were similar.

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