Fig. 4. Representative western immunoblots of post-mitochondrial supernatant either unphosphorylated or phosphorylated with HCR (heme controlled repressor kinase). (A) Helix aspersa hepatopancreas. (B) Neobatrachus sutor liver. Two identical samples from an individual were analysed, one sample was left untreated (–HCR) and the other was phosphorylated (+HCR). Samples from the same individual are shown as pairs on the figure. Proteins were separated using SDS–PAGE, transferred to nitrocellulose membranes and probed with anti-eIF2(P). The comparison between samples incubated with HCR (+HCR) and without HCR (–HCR) was used to determine the ratio of phosphorylated to unphosphorylated eIF2 in both the awake and estivating states. The bands corresponding to eIF2(P) have been quantified and are summarised in Table 1.