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. 2020 Feb 15;31(4):261–272. doi: 10.1091/mbc.E19-07-0399

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© 2020 Sébastien, Aubin, et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.

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FIGURE 2: — T95, Sec61β, and KDEL localize, respectively, in SR membranes and lumen. (A) Schematic representation of T95, Sec61β, and KDEL constructs fused to PAGFP, with N-terminal (N), C-terminal (C), and transmembrane (TM) domains sequence length and localization regarding the SR membrane (cytoplasm, membrane [Mb] and SR lumen). T95-PAGFP: TM in black, N and C-terminal in yellow and PAGFP in green; Sec61β: TM, N-, and C-terminal in blue and PAGFP in green; PAGFP-KDEL: PAGFP in green and KDEL in purple. Amino acid numbers from N-terminal to C-terminal end are indicated. (B) Western blot analysis has been performed with antibody against GFP on 10 μg triadin KO cells lysates transduced with T95-PAGFP or PAGFP-KDEL or Sec61β-PAGFP. Three bands are detected at 150 kDa (T95-PAGFP), 37 kDa (Sec61β-PAGFP), and 28 kDa (PAGFP-KDEL). The β-tubulin has been used as a loading control (bottom panel). (C) DIF3 (left) and DIF9 (right) myotubes expressing T95, Sec61β, or KDEL immunolabeled with anti-GFP (green) and anti-RyR1 (magenta) antibodies. Scale bars = 5 μm. Insets of T95, Sec61β, or KDEL immunolabelings with that of RyR1 are shown. Scale bars = 2 μm. Percentages of colocalization are shown on Figure 6D and Supplemental Figure S7 for DIF9 and DIF3, respectively .(D) Plot profiles of constructs (green) vs. RyR1 (magenta) intensities as a function of distance for 10 μm (five successive triads as shown by the yellow line on C) in TRDN KO myotubes at DIF9.