Fig. 4. In vitro phosphorylation of IP₃R1 and GST-IP₃R1 fragments by activated ERK2 kinase. (A) Purified IP₃R1 and IP₃R3 (1 µg) or GST-IP₃R1 purified fragments (0.5 µg) were phosphorylated in vitro in the presence of [-³²P]ATP at 30°C using activated ERK2. Phosphorylated IP₃Rs and GST-fusion fragments were detected using a phosphorimager. (B) Equal amounts of IP₃Rs in reaction were determined with an anti-IP₃R antibody that recognizes both isoforms equally. (C) Phosphorylation of IP₃R1 domains expressed as GST-fusion proteins by MAPK/ERK2 performed and quantified as above. Arrowheads indicate the position of ERK2 and of the various GST-fusion proteins. The amino acids included within each of the domains and the predicted molecular weight for each domain are noted in Table 2. (D) ERK2 phosphorylation of IP₃R1 domain 2 after in vitro mutagenesis of S⁴²¹ and S⁴³⁶. Equal loading was ascertained using the anti-cytI3b1 antibody (data not shown). All results are typical of at least three experiments.