Epidermal explant lysates from embryos injected into the animal hemisphere with Flag–Sdc4 mRNA and uninjected controls were diluted 1:3 in 9 M urea, pH 5.5, 1 mM EDTA, 0.1% Triton X-100, 0.2M NaCl and protease inhibitors, and were anion-exchange purified using 20 μl of DE52 gel. The released proteoglycans in 2 M ammonium bicarbonate, 0.05% Triton and BSA, which was added as a carrier, were diluted 1:10 in water and lyophilized. Equal parts of the resuspended PGs were treated with lyases. Then, equal protein amounts were separated by 8% SDS-PAGE, and immunoblotting was performed using a monoclonal anti-Intβ1 antibody (8C8, 1:140, DSHB) and HRP-conjugated anti-Flag antibody (1:1000, Sigma, A8592).
