Effects of insulin on the phosphorylation of metacestode vesicle proteins. A) Detection of EmIR1 in vesicle membrane fractions (MF). Vesicles from in vitro culture were homogenized and the MF was isolated. Western blot detection was carried out on MF and whole vesicle preparations (Ves) using the anti-EmIR1 antiserum. ‘Pro’ and ‘β’ mark the receptor pro-form and β-subunits. B) Phosphorylation of EmIR1 in response to insulin. Metacestode MF was stimulated for 10 minutes with either 100 nM insulin or IGF-I, followed by 30 minutes incubation with 100 μM HNMPA(AM)3 or control DMSO. Phosphorylation of membrane proteins was carried out for 40 minutes in the presence of [32P] γ-ATP. Proteins of the MF were precipitated using the anti-EmIR1 antiserum. Proteins were then separated by 8% SDS-PAGE and phosphorylation was detected by autoradiography. Bands are visible at the size of the EmIR1 β-subunit and below. C) Phosphorylation of EmIR1 after insulin stimulation of metacestode vesicles. Vesicles were stimulated (+) or not (−) with 100 nM insulin for 10 minutes. Following solubilisation of membrane proteins, the EmIR1 β-subunit was precipitated using the anti-EmIR1 antiserum and separated by SDS-PAGE. Western blot detection was carried out using the anti-EmIR1 antiserum (lower panel) or an anti-phospho-tyrosine antibody (upper panel). D) Phosphorylation of Echinococcus PI3K/Akt pathway components in response to insulin. Vesicles were stimulated with 10 nM insulin for the times indicated above. Vesicle lysates were subsequently separated by SDS-PAGE and probed using antibodies against the phosphorylated Akt substrate motif or phospho 4E-BP as indicated. β-Actin was used as loading control. E) Inhibition of 4E-BP phosphorylation through HNMPA(AM)3. Vesicles were incubated for two hours with 100 μM HNMPA(AM)3 (HNM+) or the PI3K inhibitor LY294002 (LY+) before stimulation with 10 nM insulin. Crude lysates were probed with the anti-phospho 4E-BP antibody. β-Actin was used as loading control. DMSO, dimethyl sulphoxide; HNMPA, 2-hydroxynaphthalen-1-yl-methylphosphonic acid.