|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 1. Illustration of GFP-b(5)tail and characterisation of HeLa TetOff cells expressing the construct. (A) Schematic representation of GFP-b(5)tail. The construct is composed of GFP fused at its C-terminus to a linker (zig-zagged line) consisting of the myc epitope followed by a repeated Gly-Ser sequence. This is attached to the entire tail region of rat cytochrome b(5), which contains the transmembrane domain flanked upstream and downstream by polar sequences. The orientation of the construct, with the N-terminal GFP in the cytosol and the C-terminus in the ER lumen is indicated. (B) Western blot analysis of GFP-b(5)tail expression in stably transfected HeLa TetOff cells. Cells were harvested at the indicated times after removal of doxycycline from the culture medium. Each lane contained 55 µg of protein. (C) Confocal analysis of HeLa TetOff cells expressing GFP-b(5)tail. Cells were fixed 3 days after exposure to doxycycline-free medium. Bar, 10 µm. (D) G-6-Pase EM cytochemistry of HeLa TetOff cells, induced (right) or not induced (left) to express GFP-b(5)tail. The product of the cytochemical reaction is found within the nuclear envelope and in ER cisternae. Note the higher density of ER cisternae in the induced cells. N, nucleus. Bar, 1 µm.
|
