Detail
| Localization |
Tissue |
Validated Method |
PMID |
| Endoplasmic reticulum |
COS-7 cell |
Fluorescence in situ hybridization |
22679391 |
|
Description: Figure 2: ALPP and CALR, but not t-ftz or INSL3, mRNA remain associated with the ER independently of ribosomes and translation. (A鈥æˆ) COS-7 cells were transfected with plasmids containing either the t-ftz (A), INSL3 (A鈥æƒ), ALPP (A, C), cyto-ALPP (a version of ALPP lacking signal sequence and transmembrane domain coding regions; A, D鈥æˆ), or CALR (A) genes and allowed to express mRNA for 18鈥?4 h. The cells were then treated with DMSO (Cont), puromycin, or HHT for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for mRNA using specific FISH probes, and imaged (see panels Béˆ¥æ‡ for examples). The fluorescence intensities of mRNA in the ER and nucleus in the micrographs were quantified (A). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. Note that although ribosome disruption caused INSL3 mRNA to dissociate from the ER, the nuclear mRNA was unaffected (B, nuclei are denoted by arrows). (E) A single field of view containing a single HHT-treated, digitonin-extracted, COS-7 cell expressing cyto-ALPP mRNA. cyto-ALPP mRNA was visualized by FISH and for Trap伪 protein by immunofluorescence. Note the extensive co-localization of cyto-ALPP mRNA (red) and Trap伪 (green) in the overlay. All scale bars?=?20 um. Data are collected from Figure 2.
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Other Subcellular Localization: