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Fig. 1. Localization of HS6ST-1, -2, and -3 in the trans-Golgi cisternae of CHO-K1 cells. (A) Schematic representation of the C-terminally GFP-tagged murine HS6ST-1, -2, and -3 proteins (m6ST1-EGFP, m6ST2-EGFP, and m6ST3-EGFP). Y denotes the N-glycosylation sites. The pink and blue boxes and the green ovals indicate the stem domain, the PAPS-binding domains and the EGFP protein, respectively. The numbers shown below indicate the amino acid number in the cytoplasmic, transmembrane, lumenal, linker, and EGFP domains. The numbers in parentheses indicate the amino acid number in the stem domains that we defined. The black arrows indicate the position of the conserved Glu-Tyr residues that are detected as the amino-termini of the HS6STs secreted in the culture medium. TM, transmembrane region. (B) Intracellular co-localization of m6ST1-EGFP, m6ST2-EGFP, and m6ST3-EGFP with the Golgi marker GM130. Cells transfected with plasmids encoding m6ST1-EGFP, m6ST2-EGFP, or m6ST3-EGFP were fixed and stained with rabbit polyclonal anti-GM130 antiserum followed by Alexa Fluor 594 goat anti-rabbit IgG secondary antibodies. From left to right, the panels show phase contrast, GM130 immunofluorescence, GFP tag fluorescence, and merged images. (C) CHO-K1 cells transfected with m6ST1-EGFP were pretreated with 100 µM cycloheximide for 30 minutes, then treated with 10 µg/ml BFA for 1 hour or 20 µM nocodazole for 1 or 2 hours in the presence of cycloheximide. Fixation and staining proceeded as in Fig. 1B.
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