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

Total cellular RNA from the cell lines was isolated according to the method of Chomczynski and Sacchi (1987) . 25 g of RNA from each cell line was denatured in 1 ϫ MOPS buffer, 0.66 M formaldehyde, and 50% (vol/vol) formamide, and subsequently size fractionated on a 1.3% agarose-formaldehyde gel. The RNA was transferred and immobilized onto Hybond-N membrane (Amersham Intl., Little Chalfont, UK). The membrane was hybridized with a 1.2-kb HindIII/XbaI cDNA fragment from the ␣ 2,3 SAT (O) plasmid according to the method of Church and Gilbert (1984) and washed to highest stringency as described previously (Brockhausen et al., 1995) . To assess the efficiency of loading and transfer of the RNA, the membrane was reprobed for 18S expression. For detecting the overexpressed transfected ␣ 2,3 SAT (O), the hybridized blot was exposed to film overnight. For detection of endogenous transcripts, blots were exposed for 6 d.