Figure 7

Insulin3 is predominantly localised in the endoplasmic reticulum.

(a) mRNAs were injected into the animal pole of both blastomeres at the two-cell stage in X. laevis embryos. Amounts of mRNA injected per embryos were: insulin3-HA (500 pg), CAAX-GFP (250 pg) and sec61β-GFP (250 pg). CAAX-GFP and Sec61β-GFP are localised at the plasma membrane and endoplasmic reticulum, respectively. Embryos were fixed at stage 11. Immunohistochemistry was performed using antibodies against HA-tag and GFP. Animal cap regions were used for analysis. Insulin3-HA was predominantly co-localised with Sec61β-GFP at the endoplasmic reticulum. (b) One ng of mRNAs was injected into the animal pole of both blastomeres at the two-cell stage in X. laevis embryos. Blastocoel fluids were collected at stage 10.5. One μL of the blastocoel fluid was used for western blotting. The lysate was equivalent to one embryo. Insulin3-HA was secreted into the blastocoel at lower efficiency than other insulin family members. Full-length blots are presented in Supplementary figure S9. (c) mRNAs were injected into the animal pole of one blastomere at the two-cell stage in X. laevis embryos. Amounts of mRNA injected per embryos were: b-gal (250 pg), dkk1 (500 pg) and insulin3-HA (500 pg). Anterior view of stage 20 embryos. Red-gal staining indicated the injected-side. The effect of insulin3-HA was in a cell-autonomous manner, unlike dkk1.