loped by chemiluminescence or detected with IRDye 680 and IRDye 800 Staining the jelly canal The sea urchin egg jelly canal was stained using a procedure adapted from Maruyama et al.. Spawned eggs were collected “dry” by placing the female urchin with the aboral surface facing down on a Petri dish. These eggs were then transferred directly into a small amount of Sumi ink solution. Following incubation in the Sumi 12 Disheveled Regulation in the Vegetal Egg Cortex secondary antibodies respectively using a LI-COR Odyssey Infrared Imaging System. 2D Western blot analysis of unfertilized eggs, 16-cell stage embryos, and micromeres was carried out using the following procedure. Samples were collected in a 1.5 ml Eppendorf tube and centrifuged at 86 x g for one minute to collect a tight pellet. The seawater was then decanted using a pipette to remove as much seawater as possible. Isolated egg cortices were collected as described above. Pelleted samples were lysed in 100 l of solubilizing solution. The protein concentrations were determined using the Bradford assay and then the samples were desalted using the ReadyPrep 2D cleanup kit. For 2D SDS-PAGE 40 g of total protein of each sample was solubilized in 125 l of 2D electrophoresis buffer ASB-14, 40 mM Tris, 0.001% Bromophenol Blue, BIO-RAD) with the addition of 25 mM DTT, 4% CHAPS, and 0.2% Bio-Lyte pH 3-10/4-7. Samples were loaded on an immobilized pH gradient ReadyStrips 4-7 and isoelectrofocused with the Protean IEF cell by applying a total of 10,000 V-hr according to the ReadyStrip IPG 21821695 Instruction Manual. The IPG strips were then equilibrated in SDS-PAGE equilibration buffer DTT) two times and transferred to a 10% Tris-Glycine Polyacrylamide Gel. SDS-PAGE was performed and the proteins were transferred onto Trans-Blot nitrocellulose membranes. Immunoblots were probed with the antiSUDshDIX and anti-tubulin antibodies as described earlier. hybridization detection of Dsh mRNA in early stage embryos shows that Dsh is ubiquitously expressed starting in the unfertilized egg to the 60-cell stage embryo. Dsh protein is expressed at different developmental stages. Tubulin serves as the loading control. Western blot analysis of isolated animal and vegetal halves from 16-cell stage embryos shows Dsh is expressed in both halves. Actin serves as the loading control. All the 
samples used in these experiments were collected from S. purpuratus. Cytoskeleton disruption studies To determine if specific components of the cytoskeleton are involved in localizing 14522929 or tethering Dsh to the vegetal cortex of the egg, specific cytoskeletal components were disrupted using the following chemicals: cytochalasin B, cytochalasin D, and colchicine . All the inhibitor stock solutions used dimethyl sulfoxide as the MedChemExpress 1235481-90-9 solution vehicle, and controls included DMSO at the same concentrations expected in the diluted inhibitor solutions used in the assays. Eggs were dejellied prior to applying the respective inhibitors. Treated eggs were incubated at 15C and samples were collected for immunostaining and Western blot analysis after 2 hours following drug treatment. Recovery experiments were done by exposing eggs to the drugs for 20 minutes, washing the eggs three times with FSW, followed by incubation of the eggs for an additional 100 minutes at 15C. To determine the timing of Dsh degradation following disruption of microfilaments, unfertilized eggs were treated with cytochalasin B or D for 5 minutes, rinsed three times in ASW, an
