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Results: 7

1.
Figure 5

Figure 5. From: GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.

Ii is sialylated in BFA-treated cells and in cells overexpressing wt rab6 and rab6 Q72L. HeLa cells were transfected, pulsed, and chased as described in the legend to Fig. 4. After immunoprecipitation, Ii immunoprecipitates were treated with (+) or without (−) neuraminidase. Asterisks (∗) point to the shift in mobility of Ii caused by the removal of sialic acids. Note that both p31 and p33 are shifted down upon neuraminidase treatment.

Olivier Martinez, et al. Proc Natl Acad Sci U S A. 1997 March 4;94(5):1828-1833.
2.
Figure 7

Figure 7. From: GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.

rab6 Q72L-induced redistribution of gal-T is microtubule-dependent. HeLa cells were cotransfected with Ii and either pGEM-1 (AD) or rab6 Q72L (E and F) plasmids. Nocodazole (10 μM; B, D, and F) and BFA (5 μg/ml; C and D) were added to the cells 3 and 4 h posttransfection, respectively. Cells were fixed 6 h posttransfection with 4% paraformaldehyde and double labeled with anti-Ii and anti-gal-T antibodies. We only show in this figure gal-T staining. All of these cells were also stained with anti-Ii antibody.

Olivier Martinez, et al. Proc Natl Acad Sci U S A. 1997 March 4;94(5):1828-1833.
3.
Figure 2

Figure 2. From: GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.

Colocalization of gal-T and Ii in rab6 Q72L transfected cells. HeLa cells were cotransfected with Ii and either pGEM-1 (A) or rab6 Q72L (B) plasmids. Cells were fixed 6 h after transfection and processed for cryosectioning and immuno-electron microscopy. Cells were double labeled with antibody against gal-T and anti-Ii antibody. Primary antibodies were visualized with protein A-gold conjugates (gal-T, 10 nm; Ii, 5 nm). In control cells (A), anti-gal-T antibody labels typical Golgi stacks (g). Arrowheads in A point to Ii molecules localized in Golgi stacks in control cells. Arrows in B point to gal-T molecules localized within ER cisternae (Ii-positive) in rab6 Q72L transfected cells. (Bars = 0.1 μm.)

Olivier Martinez, et al. Proc Natl Acad Sci U S A. 1997 March 4;94(5):1828-1833.
4.
Figure 1

Figure 1. From: GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.

Overexpression of rab6 Q72L promotes the redistribution of Golgi gal-T into the ER. HeLa cells cotransfected with Ii and either pGEM-1 (AD), rab6 Q72L (E and F), or rab6 T27N (G and H) plasmids were fixed 6 h after transfection with 4% paraformaldehyde and processed for immunofluorescence. BFA (5 μg/ml) was added 2 h before fixation (C and D). After permeabilization with saponin, cells were double labeled with a monoclonal antibody against Ii (A, C, E, and G) and a polyclonal antibody against gal-T (B, D, F, and H). The arrows in E and F point to a nontransfected cell that displays normal gal-T staining. All pictures shown here represent stacks of four medial optical slices obtained by confocal microscopy and separated from each other by 0.5 μm.

Olivier Martinez, et al. Proc Natl Acad Sci U S A. 1997 March 4;94(5):1828-1833.
5.
Figure 6

Figure 6. From: GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.

(A) Ii progressively acquires O-linked sugars in control cells. HeLa cells cotransfected with Ii and empty pGEM-1 plasmids were metabolically labeled for 10 min and chased for 0, 1, or 3 h. Ii immune precipitates were used in the jacalin binding assay as described in the legend to Fig. 4. Radioactivity in each sample (total and jacalin-bound) was directly measured with the β counter. Error bars represent the range in three independent experiments. (B) rab6 T27N inhibits O-glycosylation of Ii. HeLa cells cotransfected with Ii and either pGEM-1 (control) or rab6 T27N (T27N) plasmids were metabolically labeled for 10 min and chased for 3 h. Ii bound to jacalin was calculated as described above. Error bars represent the range in three independent experiments. The data in A and B are shown after subtraction of background (counts obtained after 10-min pulse).

Olivier Martinez, et al. Proc Natl Acad Sci U S A. 1997 March 4;94(5):1828-1833.
6.
Figure 4

Figure 4. From: GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.

rab6 Q72L, wt rab6, and BFA increase O-glycosylation of Ii. HeLa cells cotransfected with Ii and either empty plasmid (control, BFA) or the indicated rab6 constructs (WT, Q72L, T27N) were metabolically labeled for 10 min and chased for 3 h. In BFA-treated cells, 5 μg/ml BFA was added to the chase medium. Ii immune precipitates were solubilized with SDS and nine-tenths of each eluate was diluted in Triton X-100 containing buffer and incubated with biotinylated jacalin and streptavidin-agarose. O-glycosylated Ii bound to jacalin was specifically eluted with galactose. (A) Visualization by autoradiography of Ii present in the different cell lysates before (Total) or after binding to jacalin (Jacalin). Total represents one-tenth of the whole immunoprecipitate for each sample. (B) Ii bands were quantified by scanning with the PhosphorImager. This graph represents the means ± SD of two independent experiments.

Olivier Martinez, et al. Proc Natl Acad Sci U S A. 1997 March 4;94(5):1828-1833.
7.
Figure 3

Figure 3. From: GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.

rab6 Q72L and BFA induce posttranslational modifications of Ii that do not correspond to a maturation of N-linked sugars. HeLa cells cotransfected with Ii and either pGEM-1 (control, BFA) or rab6 Q72L (Q72L) plasmids were metabolically labeled for 10 min with [35S]methionine and [35S]cysteine and chased in medium alone (control, Q72L) or medium containing 5 μg/ml BFA. Ii present in cells was immunoprecipitated after 3 h. Immune precipitates were then divided into three equal parts and treated with or without endo H or N-glycanase (N-gly). Human Ii cDNA contains two ATGs, which generate two forms of Ii, Iip31 and Iip33 (arrows). Ii present in rab6 Q72L overexpressing cells and in BFA-treated cells displayed the same shift in mobility as compared with control cells (tracks undigested). In all cell lysates, Ii was endo H-sensitive (tracks endo H). The treatment with N-glycanase did not abolish the difference in mobility seen between Ii from control cells and Ii from BFA-treated or rab6 Q72L transfected cells.

Olivier Martinez, et al. Proc Natl Acad Sci U S A. 1997 March 4;94(5):1828-1833.

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