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. 2018 Jan 12;8:677. doi: 10.1038/s41598-017-19130-y

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© The Author(s) 2018

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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Lysosome sorting of ADRB2_CIB after prolonged irradiation. (a) Images of ADRB2_CIB and lysosome marker LAMP1 fluorescence in HEK293_opt cells before and after blue light irradiation for 120 min. The white arrowheads indicate the colocalization of ADRB2_CIB and LAMP1. Red, ADRB2_CIB; green, LAMP1. Bar, 20 μm. (b) Temporal changes in the colocalization of ADRB2_CIB with lysosomes in HEK293_opt cells stimulated with blue light for the indicated time or with ISO for 120 min. Data are shown as the mean ± s.e.m. (n = 9 from two independent experiments) The statistical analysis was performed unpaired Student’s t-test (two-tailed). **P < 0.01 (P = 2.0 × 10⁻⁷). (c) IP-Western blotting analysis for the detection of ubiquitinated ADRB2_CIB. HEK293_opt cells were stimulated with blue light for the indicated time or with 1.0 μM ISO for 120 min. ADRB2_CIB was immunoprecipitated using an anti-V5-tag antibody. Ubiquitin, ADRB2 and β-actin were blotted with antibodies specific to them. (d) The quantified temporal changes in ubiquitinated ADRB2_CIB were calculated as the ratio of ubiquitinated ADRB2_CIB to total ADRB2_CIB quantities, which were determined in the Western blot analysis. The ratio values were normalized to their value at 0 min. Bars: mean ± s.e.m. (n = 9 from three independent experiments). The statistical analysis was performed unpaired Student’s t-test (two-tailed). **P < 0.01 (P = 1.1 × 10⁻⁴).