Fig. 1. The recombinant MAP1B protein SP contains the GSK-3ß phosphorylation site recognised by mAb SMI-31, whereas SP does not. (A) Diagram showing full-length mouse MAP1B and the two MAP1B recombinant proteins (SP and SP) used in the kinase assay. The SPAKS site is shown as a hatched box and its sequence, beneath the full-length MAP1B, in the single letter amino acid code. One recombinant protein (SP) encodes amino acids 1244-1530, and hence starts at the beginning of the SPAKS sequence, whereas the other (SP) lacks the SPAKS sequence by starting at amino acid 1264. GST, glutathione S-transferase. (B) Western immunoblots of SP and SP GST-pull downs that were incubated with (+), or without (–), a high-speed supernatant from neonatal rat brain (S1) in kinase buffer showed that SP was positive for the mAb SMI-31 epitope, whereas SP was negative. The same blot was then stripped and re-probed with an anti-GST antibody (GST) to assess the protein loading levels, which were found to be similar (lower panel). Note that both recombinants, when incubated with S1, show an upward band shift. This is normally associated with protein phosphorylation, and suggests, therefore, that the SP recombinant becomes phosphorylated in the kinase assay, but at a site or sites not recognised by mAb SMI-31.