Fig. 1.

Endogenous Sec61b and nesprin-2 mRNA associates with the ER membrane. U2OS cells were either: fixed (Unextracted); first extracted with digitonin and then fixed (Extracted); or pre-treated with puromycin (Puro) or homoharringtonine (HHT) for 30 min, extracted with digitonin in the presence or absence of EDTA and then fixed. Cells were stained with a pool of FISH probes to visualize individual endogenous human Sec61b, nesprin-2 or GAPDH mRNA molecules. Each cell was visualized by phase microscopy to determine the cell contours. mRNA foci were identified using the NIS-element ‘Spot Detection’ function (see Materials and Methods section). (A) mRNA FISH signals overlaid with the contours of the cells and nuclei, and with the detected foci highlighted by the spot detection function. (B) The number of cytoplasmic (i.e. non-nuclear) foci were determined for each condition. Each bar is the mean±s.e.m. of 30 cells. Scale bar: 20 µm.