Fig. 1. Signal-dependent phosphorylation of Pelle during early embryogenesis. (A) Pelle is transiently modified in early embryos. Whole embryo extract was prepared from wild-type embryos of different stages (see Materials and Methods), and equivalent amounts of extract were subjected to SDS-PAGE. Pelle was detected by anti-Pelle immunoblotting (top). Anti-Toll immunoblotting was used as control (bottom). Lanes 1-6, embryo extracts were from 1- to 6-hour-old embryos as indicated. (B) Pelle modification is enhanced in gain-of-function Toll mutant embryos. Wild-type or mutant embryos with an activated Toll allele (Toll^10b) were collected and analyzed as in A. The top panel is an anti-Pelle blot, and the bottom panel is a blot with anti-Toll antiserum. Lanes 1-5, wild-type embryos between 0 and 5 hours after egg laying; lanes 6-10, Toll^10b embryos between 0 and 5 hours after egg laying. (C) Pelle modification is decreased in loss-of-function Toll mutant embryos. Wild-type, Toll^10b, gastrulation defective null mutant (Gd^–/–), weak loss-of-function Toll mutant (Tollr444) and Toll knockout (9QRX) embryos were collected 2-4 hours after egg laying and analyzed by SDS-PAGE and western blot as in A. (D) Pelle is phosphorylated in Toll^10b embryos. For dephosphorylation by endogenous phosphatases, Toll^10b whole embryo extract from the second to fourth hours after egg laying (lane 1) was incubated at 30°C with or without NaF (lane 2, 3) for 1 hour. The same extract was also incubated with immobilized calf intestine phosphatase (CIP-beads), with or without NaF (lane 4, 5), at 37°C for 1 hour. Pelle protein was analyzed by SDS-PAGE followed by anti-Pelle immunoblotting.

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