Journal: The Journal of Cell Biology
Article Title: Assembly and trafficking of caveolar domains in the cell
Figure Lengend Snippet: Cav1-containing structures move long distances in activated cells. (A) Large peripheral areas of CV-1 cells expressing Cav1-GFP were bleached (marked areas in bleach panels), and the movement of Cav1-GFP into the bleached area was monitored omitting the 5-μm region closest to the bleach boundary (marked in 15 min panels). The experiment was performed in the absence (untreated, upper panels) and the presence of SV40 (1 h incubation, MOI 60; +SV40, lower panels). Before (Prebleach), immediately after (Bleach), and 15 min after (15 min) bleaching are shown. Note the increase in long-distance movement in the presence of SV40 (see Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1 ). Bars, 10 μm. (B) Recovery of fluorescence due to the long-distance movement of Cav1-GFP in CV-1 cells increases after addition of SV40, vanadate, or latA. The CV-1 cells expressing Cav1-GFP were either untreated, exposed to SV40 (MOI 60) for 1 h, to 1 mM vanadate for 1 h, to 5 μM nocodazole for 30 min, to 5 μM nocodazole for 30 min and then 1 h to SV40 (MOI 60), or to 0.8 μM latA for 10 min before the FRAP experiments. The fluorescence recovery was quantified in the bleached area omitting the 5 μm region closest to the bleach boundary (marked in 15 min panels in A) after 15 min. Recovery was calculated by measuring the fluorescence intensity in the defined area before and 15 min after bleaching (see Videos 5–7). The error bars indicate standard deviations of five independent experiments.
Article Snippet: To see the effect of ligand and drugs, the cells were treated as follows: infected with SV40 (MOI 60) for 1 h, treated with 1 mM sodium orthovanadate (vanadate; Calbiochem-Novabiochem) for 1 h, 100 μM genistein (Sigma-Aldrich) for 30 min, and then incubated further 1 h with SV40 (MOI 60), treated with 5 μM nocodazole (Sigma-Aldrich) for 30 min, treated with 5 μM nocodazole for 30 min, and then incubated with SV40 (MOI 60) for 1 h, or treated with 0.8 μM latA (Molecular Probes) for 10 min. A defined region was bleached at full laser power (100% power, 100% transmission, 25 iterations) using the 488 nm line from a 30 mW Argon/2 laser.
Techniques: Expressing, Incubation, Fluorescence