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. 2021 Sep 16;184(19):4886–4903.e21. doi: 10.1016/j.cell.2021.08.001

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© 2021 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Figure S3 — Additional multi color stainings of different OT film-like organelle constructs with organelle markers, related to Figure 4

(A–D) HEK293T cells expressing the indicated OT organelle, tRNA^Pyl and the double-recruitment reporter (EGFP^39TAG::boxB, mCherry^190TAG::ms2) in the presence of the ncAA SCO-K. All images show a selected plane of a z stack. Scale bars: 20 μm.

(A) IF staining against PylRS and a plasma membrane marker (NaK-ATPase). Top to bottom: PylRS (magenta), NaK-ATPase (cyan), NLS::EGFP^39TAG::boxB (blue), NLS::mCherry^190TAG::ms2 (yellow) and merged.

(B) IF staining against PylRS and a Golgi membrane marker (Giantin). Top to bottom: PylRS (magenta), Giantin (cyan), NLS::EGFP^39TAG::boxB (blue), NLS::mCherry^190TAG::ms2 (yellow) and merged.

(C) IF staining against PylRS and an ER membrane marker (Calnexin). Top to bottom: PylRS (magenta), Calnexin (cyan), NLS::EGFP^39TAG::boxB (blue), NLS::mCherry^190TAG::ms2 (yellow) and merged.

(D) IF staining against PylRS and a mitochondrial marker (apoptosis-inducing factor, AIF). Top to bottom: PylRS (magenta), AIF (cyan), NLS::EGFP^39TAG::boxB (blue), NLS::mCherry^190TAG::ms2 (yellow) and merged.

Note that we do not have 100% transfection efficiency, while all the used markers are endogenous to the cell.