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. 2021 Sep 1;12:5204. doi: 10.1038/s41467-021-25546-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic illustration for secretory protein labeling by ER-localized TurboID (TurboID-KDEL) or ER-anchored TurboID (Sec61b-TurboID). b Immunofluorescence localization of TurboID (Anti-V5) and biotinylated proteins (Streptavidin-Alexa) in HeLa cells transfected with TurboID-V5-NES or TurboID-V5-KDEL or Sec61b-V5-TurboID expression plasmids. Scale bars, 10 μm. c Western blots for biotinylated proteins (Streptavidin-HRP) and TurboID (Anti-V5) in cell lysates or culture supernatant of NIH-3T3 cells transfected with GFP (Control), TurboID-V5-KDEL (KDEL), or Sec61b-V5-TurboID (Sec61b) expression plasmids. Anti-GAPDH as a loading control. Asterisk indicates self-biotinylated TurboID-KDEL or Sec61b-TurboID. d Line-scan analysis of biotinylated proteins in cell lysate (orange) or culture supernatant (black) from NIH-3T3 cells transfected with Sec61b-V5-TurboID expression plasmids and treated with biotin. PC, Pyruvate carboxylase; MCC/PCC, Methylcrotonyl-CoA Carboxylase/ Propionyl-CoA carboxylase. e Effect of Brefeldin A (BFA) on biotinylated protein secretion in HepG2 cells transfected with Sec61b-V5-TurboID expression plasmids. f Time course blot for biotinylated-labeled proteins (Streptavidin-HRP) in cell lysates of HepG2 cells transfected with Sec61b-V5-TurboID expression plasmids. g Time course blot for biotinylated protein (Streptavidin-HRP) turnover in cell lysates of HepG2 cells transfected with Sec61b-V5-TurboID expression plasmids following biotin washout. h Quantitation and plotting of the time course blot for biotinylated protein turnover shown in g. Asterisk indicates Sec61b-TurboID. Source data for c–h are provided as a Source Data file. These experiments were repeated as biological triplicates with similar results.