Supersignal signal west pico plus

For each sample, five adult Drosophila heads were sonicated in SDS sample buffer (50 mM Tris–HCl, ph 6.8, 10% SDS, glycerol, Dithiothreitol (DTT), bromophenol blue); N refers to the number of samples tested. Proteins were separated on a 10% acrylamide gel. Nitrocellulose blots were probed with primary antibodies overnight at room temperature: anti-Kv4/Shal 1:100, anti-actin 1:10000 (EMD Millipore), anti-GFP 1:10000 (Torrey Pines Biolabs); anti-HA 1:500 (Covance Research Products), and anti-syntaxin 1:50 (Developmental Hybridoma Studies Bank). Anti-K_v4/Shal antibodies were generated as previously described (Diao et al., 2010 (link), 2009 (link)). Blots were incubated with peroxidase-conjugated secondary antibodies (1:1000; Jackson ImmunoResearch Laboratories) for one hour at room temperature, developed using Supersignal Signal™ West Pico PLUS (Thermo Scientific). K_v4/Shal, GFP, and HA signal densities were normalized to densities from loading control signals from the same lane.