Chemiluminescence kit

A rabbit polyclonal antibody raised against amino acids 110-335 of the human ADHFe1 (PA5-31416; Thermo Fisher Scientific) was used to detect the Xenopus laevis protein. Protein lysates for Western blots were prepared by homogenizing embryos or HEK293 cells in ice-cold lysis buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with protease inhibitors (1 mM phenylmethyl sulfonyl fluoride, 1 mM pepstatin, and 10 mg/ml aprotinin). Homogenates were cleared by centrifugation at 14,000 rpm for 10 min at 4°C. Sodium dodecyl sulfate (SDS) sample buffer was added to the cleared lysate and boiled for 4 min before separation by SDS-polyacrylamide gel electrophoresis. Variability in cell densities was minimized by normalization to total protein concentration measured with the protein assay kit (Bio-Rad). Proteins were blotted to polyvinylidene difluoride membranes (Bio-Rad), and blots were blocked in 5% milk in TBS + 0.1% Tween and probed with anti-ADHFe1 antibody (1:10000) for 1 hr at room temperature and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20000; A0545, Sigma) as a second antibody. Immunoreactive proteins were detected using a chemiluminescence kit (Biological Industries) according to the manufacturer's protocol.