Larval Brain Western Blot Analysis

Larval brains from wandering third instar larvae were dissected and transferred into 2× sample buffer (Life Technologies). NuPage gels (Invitrogen) were loaded with five brains per lane. Western blotting was performed according to Enneking et al. (2013) (link). In brief, membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti-Nrg^3C1, which recognizes extracellular domains of Nrg¹⁸⁰ and Nrg¹⁶⁷ (1:500; a gift from M. Hortsch, University of Michigan, Ann Arbor, MI; Hortsch et al., 1990 (link)); mouse anti-Nrg¹⁸⁰ (BP104; 1:200; Hortsch et al., 1990 (link)); and mouse anti–β-tubulin (E7; 1:50; both from the Developmental Studies Hybridoma Bank) followed by a 2-h incubation at room temperature with secondary HRP-conjugated goat anti–mouse and goat anti–rabbit at 1:10,000 (Jackson ImmunoResearch Laboratories, Inc.).