Figure 7.

Autophosphorylation by GST-tagged mutant proteins varies greatly. Plasmids carrying the GST-tagged mps1 alleles were transformed into the wild-type strain FLY14A (Table 1), and expression of fusion proteins was induced as described (see MATERIALS AND METHODS). (A) GST fusions were detected on Western blots of whole cell lysates with anti-GST antibody. The control lane (pEG) shows cells carrying the GST vector without insert. The tagged wild-type protein (GM-WT) is phosphorylated, causing it to migrate above its predicted molecular weight of 112 kDa (Lauzéet al., 1995). (B) GST fusions were affinity-purified with glutathione-Sepharose and used for in vitro kinase assays at 25°C. Material was then separated on an SDS-PAGE gel and blotted. Proteins loadings are approximately equal across the gel, as determined by Western blot (our unpublished results).