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Fig. 5. The phosphorylation sites recognised by pAb BUGS and pAb SuperBUGS are generated by GSK-3ß phosphorylation. Western immunoblot analysis of the SP recombinant protein incubated with a high-speed supernatant from neonatal rat brain (S1) in kinase buffer (+). Pre-treatment of S1 with 20 mM lithium chloride (Li) considerably reduced the binding of pAb BUGS and pAb SuperBUGS to the SP recombinant suggesting that GSK-3ß phosphorylation is necessary for these antibodies to recognise the SP protein. 20 mM sodium chloride (Na) was added to control for possible ion and concentration effects of lithium chloride. The same blots were then stripped and re-probed with an anti-GST antibody (GST) to assess the protein loading levels, which were similar. Note that band-shifts of SP produced by S1 incubation are reduced by lithium but not abolished, suggesting that SP is additionally phosphorylated by kinases other than GSK-3ß. The last track shows that baculovirus produced recombinant GSK-3ß can phosphorylate SP at the pAb BUGS and the pAb SuperBUGS site.





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