Confluent cells (CHO Fut2 simple transfectants, CHO Fut2/A double transfectants, and CHO Fut2/A/PS triple transfectants) were rinsed with ice-cold PBS, pH 7.2, and then recovered by scraping. After washing with ice-cold PBS, cells were solubilized in 50 mM potassium phosphate, pH 6.0, containing 2% (v/v) triton X-100 on ice for 30 min. Following a centrifugation at 13,000 × g for 10 min, the supernatant was collected and the protein concentration was determined using the BC assay protein quantification kit (Uptima, Montluçon, France). Thirty micrograms of total proteins of each extract were separated on 8% SDS–PAGE under reducing conditions and electrotransferred to immobilon P sheets (Millipore, Bedford, MA). Immunoblots were saturated for 1 h at room temperature with Western blocking reagent (Roche Diagnostics GmBH, Mannheim, Germany) and strips were cut and incubated overnight at 4°C with the anti-A 3-3A mAb at 2 μg/mL in antibody Western blocking reagent. Following three washes for 15 min with TBS containing 0.05% Tween 20, strips were incubated with horseradish peroxidase-labeled anti-mouse IgG (H + L) (Beckman Coulter, Fullerton, CA) for 1 h at room temperature. After three final washes, detection was performed with a chemiluminescence kit (Roche Diagnostics).