A Transfected Sialyltransferase That Is Elevated in Breast Cancer and Localizes to the medial/trans-Golgi Apparatus Inhibits the Development of core-2–based O-Glycans

Whitehouse, Caroline; Burchell, Joy; Gschmeissner, Stephen; Brockhausen, Inka; Lloyd, Kenneth O.; Taylor-Papadimitriou, Joyce

Confluent cell cultures were washed with cold PBS and lysed in RIPA buffer (20 mM sodium phosphate, pH 7.2, 50 mM sodium fluoride, 5 mM EDTA, 1% Triton, 1% deoxycholate). After clarification of the lysates by centrifugation at 15,000 g for 10 min at 4 Њ C, the protein concentration of the lysates was estimated using the Bio-Rad protein assay kit (Bio Rad Laboratories, Hercules, CA). Samples equivalent to 50 g were electrophoretically separated on a 5-15% gradient/3% stacking SDS-PAGE gel and transferred onto Hybond-C membrane (Amersham Intl.). Immunoblots were blocked with 5% skimmed milk/0.1% Tween-20 in PBS for 2 h, incubated with 0.7 g/ml anti-myc mAb, 9E10, for 1 h, and rinsed in 1% skimmed milk/0.1% Tween followed by peroxidase-conjugated rabbit antimouse secondary antibody (Dako Ltd., High Wycombe, UK) for 1 h. The bands were visualized using the enhanced chemiluminescence detection kit (Amersham Intl.).