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. 2023 Apr 12;616(7958):764–773. doi: 10.1038/s41586-023-05927-7

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 3 — a. Representative images of HEK-293T cells transfected with the cytosolic BioID2 construct and then treated with either biotin in PBS or solely PBS (no biotin). The cells were immunostained with both anti-HA antibody (red) and a fluorophore conjugated streptavidin probe (green). b. Quantification of HA intensity and biotin intensity (arbitrary units) in cells transfected with cytosolic BioID2. Black horizontal line depicts the mean, red lines depict the SEM (n = 11 cells in the no biotin group and n = 21 cells in the biotin group from 4 transfections; two-tailed unpaired t-test, P = 0.97 and P = 0.0001, respectively). c. Representative western blot of HEK-293T cells transfected with cytosolic BioID2 and treated with either biotin in PBS or solely PBS (no biotin). d. Western blot quantification of HEK293T cells transfected with cytosolic BioID2 and treated with either biotin in PBS or solely PBS (no biotin). Graph depicting the streptavidin signal intensity divided by the β-actin signal intensity for each data point. Black horizontal line depicts the mean, red lines depict the SEM (n = 8 coverslips from 3 transfections; two-tailed unpaired t-test, P = 0.0062). e. Representative images of HEK-293T cells transfected with the plasma membarane Lck-BioID2 construct and then treated with either biotin in PBS or solely PBS (no biotin). The cells were immunostained with both anti-HA antibody (red) and a fluorophore conjugated streptavidin probe (green). Arrows show the plasma membrane localization. f. Quantification of HA intensity and biotin intensity (arbitrary units) in cells transfected with Lck-BioID2. Black horizontal line depicts the mean, red lines depict the SEM (n = 8 cells in the no biotin group and n = 21 cells in the biotin group from 4 transfections; two-tailed unpaired t-test, P = 0.91 and P = 0.0084, respectively). g. Representative western blot of HEK-293T cells transfected with Lck-BioID2 and treated with either biotin in PBS or solely PBS (no biotin). h. Western blot quantification of HEK293T cells transfected with Lck-BioID2 and treated with either biotin in PBS or solely PBS (no biotin). Graph depicting the streptavidin signal intensity divided by the β-actin signal intensity for each data point. Black horizontal line depicts the mean, red lines depict the SEM (n = 11 coverslips from 3 transfections; two-tailed unpaired t-test, P = 0.01). For blot source data, see Supplementary Fig. 1.