Immunoblotting for CD55 Detection

Immunoblotting was performed as previously described (16 (link), 17 (link)), except that cell lysates or membrane fractions were analyzed using nonreducing (-DTT) SDS-PAGE for detecting CD55. Blotted polyvinylidene fluoride membranes (Millipore) were blocked for 1 h with 5% skim milk and 0.1% Tween 20 in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, 8.1 mM NaH₂PO₄, pH 7.4) and incubated with primary antibodies (Table S4) diluted in blocking solution. The blot was washed in 0.1% Tween 20 in PBS, followed by incubation for 1 h with 1:3000 dilutions of horseradish peroxidase–conjugated antimouse, anti-rabbit, or antirat secondary antibodies (NA931, NA934, or NA935; Cytiva). Immunoreactive bands were detected using Immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore) or SuperSignal West Femto maximum sensitivity substrate (Pierce; Thermo Fisher Scientific) according to the manufacturer’s instructions. Chemiluminescence images were obtained using ImageQuant LAS 500 (Cytiva).