Journal: Glycobiology

Article Title: Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.

doi: 10.1093/glycob/11.8.633

Figure Lengend Snippet: Fig. 7. Subcellular fractionation of HeLa (A–C) and LS 174T (D–F) cells for assay of glycosyltransferase relocalization on NH4Cl treatment. Cells were homogenized in buffered KCl by passage through syringe needles, debris was pelleted, and the obtained postnuclear supernatant was fractionated by ultracentrifugation on a linear Nycodenz gradient. The nine fractions, recovered from the top to the bottom, were subjected to tabletop ultracentrifugation, and the obtained pellets were reduced and analyzed by western blots of SDS–PAGE, assaying calnexin (A and D), GalNAc-T2/VSV-G (B), endogenous GalNAc-T2 (E) and Gal-T1 (C and F), and developed by the ECL method. In addition to images of the western blots, where the upper in every pair is from control cells and the lower from NH4Cl-treated cells, video densitometry measurements are shown. Open circles, control cells; filled-in circles, NH4Cl-treated cells. The difference in calnexin staining between control and treated cells in A, fraction 8, is not significant.

Article Snippet: The MUC2-producing colon adenocarcinoma cell line LS 174T (ATCC CL 188) (Asker et al., 1995), and monolayer HeLa cells (ATCC CCL 185), stable expressing VSV-Gtagged GalNAc-T2 (Röttger et al., 1998), myc-tagged NAGT I (Nilsson et al., 1993), and GFP-tagged KDEL receptor, were cultivated as described.

Techniques: Fractionation, Western Blot, SDS Page, Control, Staining

Journal: Glycobiology

Article Title: Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.

doi: 10.1093/glycob/11.8.633

Figure Lengend Snippet: Fig. 8. Effect of NH4Cl treatment on the Golgi structure in LS 174T cells. Immuno-EM of ultrathin cryosections of control cells (A) and cells treated with 25 mM NH4Cl for 40 h (B). Bar, 200 nm.

Article Snippet: The MUC2-producing colon adenocarcinoma cell line LS 174T (ATCC CL 188) (Asker et al., 1995), and monolayer HeLa cells (ATCC CCL 185), stable expressing VSV-Gtagged GalNAc-T2 (Röttger et al., 1998), myc-tagged NAGT I (Nilsson et al., 1993), and GFP-tagged KDEL receptor, were cultivated as described.

Techniques: Control

Journal: Glycobiology

Article Title: Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.

doi: 10.1093/glycob/11.8.633

Figure Lengend Snippet: Fig. 9. Gas chromatograms of released mucin oligosaccharides. O-linked oligosaccharides were released from purified insoluble mucins (MUC2) of LS 174T cells by β-elimination and fractionated into neutral, sialylated, and sulfated fractions. Gas chromatograms of permethylated neutral (A and C) and sialylated (B and D) oligosaccharides released from mucins of control (A and B) and NH4Cl-treated (C and D) cells. Designations on the peaks refer to Figure 10.

Article Snippet: The MUC2-producing colon adenocarcinoma cell line LS 174T (ATCC CL 188) (Asker et al., 1995), and monolayer HeLa cells (ATCC CCL 185), stable expressing VSV-Gtagged GalNAc-T2 (Röttger et al., 1998), myc-tagged NAGT I (Nilsson et al., 1993), and GFP-tagged KDEL receptor, were cultivated as described.

Techniques: Purification, Control