The coding potential of GFP-Sec61b is not required for its localization to the ER. (A) COS7 cells were transfected with plasmid encoding various GFP-tagged.

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The coding potential of GFP-Sec61b is not required for its localization to the ER. (A) COS7 cells were transfected with plasmid encoding various GFP-tagged tail-anchored proteins and allowed to express for 18–24 h. The coding potential of GFP-Sec61b is not required for its localization to the ER. (A) COS7 cells were transfected with plasmid encoding various GFP-tagged tail-anchored proteins and allowed to express for 18–24 h. The cells were treated with control medium or HHT for 30 min, then either directly fixed or extracted with digitonin and then fixed. Cells were stained for mRNAs using specific FISH probe against the GFP-coding sequence, which was then imaged and quantified. Fluorescent intensities in the cytoplasm and nucleus were quantified. All results were normalized to the cytoplasmic staining intensity in the unextracted cells. Each bar represents the mean±s.e.m. of three independent experiments, each consisting of at least 30 cells. (B) Hydrophobicity (y-axis, left) of the polypeptides encoded by GFP–Sec61b and GFP-fs-Sec61b was plotted against the peptide length (x-axis, bottom). Kyte–Doolittle hydropathy values were computed with ProtScale (http://web.expasy.org/protscale/), using a moving window size of 21 amino acids. Note the high hydrophobicity of the TMD region of GFP–Sec61β that is lost in GFP–fs-Sec61β. (C) COS7 cells were transfected with plasmid encoding GFP–fs-Sec61b and allowed to express mRNA for 18-–24 h. Cells were then treated with control medium or HHT for 30 min, and then either fixed (Unextracted) or extracted with digitonin and then fixed (Extracted). Cells were stained for mRNAs using a specific FISH probe against the GFP-coding sequence, and for DNA using DAPI. Each row represents a single field of view imaged for GFP–fs-Sec61b mRNA, GFP protein and DAPI. (D) Quantification of the cytoplasmic (in unextracted cells), ER (in extracted cells) and nuclear fluorescence intensities of GFP–fs-Sec61b mRNA. Each bar represents the mean±s.e.m. of 30 cells. Scale bar: 20 µm. Xianying A. Cui et al. J Cell Sci 2015;128:3398-3410 © 2015. Published by The Company of Biologists Ltd