Immunoblotting of integrin β1 and Flag-Sdc4

Cleared lysates from the epidermis-enriched explants of 12 uninjected embryos were lysed in MBS, 1% Triton X-100, 5 mM EDTA and proteinase inhibitors for 30 min on ice, centrifuged at 10,000 g for 30 min at 4°C and dialyzed with 10 mM sodium acetate pH 7.0. The protein content was measured using the Bradford method (Bio-Rad), with BSA as a standard. Equal protein amounts were treated with lyases as described above.

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).