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. 2020 Jan 2;11(1):5. doi: 10.1038/s41419-019-2199-4

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© The Author(s) 2020

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. 3 — a, b The expression levels of ALG-2 in ALG-2-OX and ALG-2-KD Jurkat cell lines. The protein levels of ALG-2 and GAPDH are determined by western blotting. c, d ALG-2 enhances the activity of proteasome in Jurkat cells. The ALG-2-OX and ALG-2-KD Jurkat cells, either inactivated or activated, were incubated in digitonin permeabilization buffer with slight sonication. Cell lysates were incubated for 1 h at 37 °C in reaction buffer with Suc-LLVY-AMC. The activity of proteasome was quantitated by AMC fluorescence intensity in a microtiter plate reader at λ^EX = 380 nm/λ^EM = 460 nm. e ALG-2 enhances the activity of proteasome in stimulated PBMCs. PBMCs were infected with ALG-2 retrovirus (left panel) or sh-ALG-2 retrovirus (right panel) for 40 h and activated by PMA and ionomycin for 10 h. Then the cells were measured the activity of proteasome as c. f ALG-2 enhances the activity of proteasome dependent on its calcium-binding sites. ALG-2-KD Jurkat cells were infected with ALG-2 WT, ALG-2 E47D, ALG-2 E114D, and ALG-2 D169A retrovirus for 48 h, then activated by PMA and ionomycin for 10 h. Proteasomal activities were assessed by Suc-LLVY-AMC cleavage as c. g Co-immunoprecipitation assays of ALG-2 mutants with Rpn3. Data shown were representative of three independent experiments. Error bars indicate SD. **P < 0.01; ***P < 0.001.