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. 2022 Jun 2;11(1):31. doi: 10.1038/s41389-022-00404-8

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© The Author(s) 2022, corrected publication 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 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. 5 — A Western bolt analysis of USP53 and CYCS proteins in HCC cell lines and L02 hepatocytes showing their relation of expression level in cell lines. B IHC staining of USP53 and CYCS in patient tissues. Scale bar:100 μm. C Spearman’s correlation analysis of USP53 and CYCS based on the IHC assay of patient samples. (n = 21). D Spearman correlation analysis of USP53 and CYCS mRNA level (LOG) in clinical samples of HCC tissues. (n = 28). E CYCS levels in USP53-overexpressing Huh-7 and HCCLM3 cells. F Western blot analysis of CYCS protein in Huh-7 and HCCLM3 cells. Cells were treated with 25 μM MG132 or CQ for 8 h before harvest. G Western blot analysis of CYCS after USP53 was knocked down in Huh-7 cells, the cells were treated with 25 μM MG132 or CQ for 8 h before harvest. H Western blot analysis showing the protein degradation time of CYCS in Huh-7 and HCCLM3 cells. I, J Western blot analysis of HA-CYCS protein level in Huh-7 and HCCLM3 cells transfected with HA-tagged CYCS plasmids and Flag-tagged USP53 plasmids or empty vectors. The transfected cells were treated with 100 μg/ml cycloheximide (CHX) for indicated time points. The results were normalized. K, L Western blot analysis of endogenous CYCS protein in Huh-7 cells with USP53 overexpressed or knocked down. The transfected cells were treated with 100 μg/ml cycloheximide (CHX) for indicated durations. Each group were repeated for three times. The results were normalized. M IP assays in HEK-293T cells, Huh-7 cells, and HCCLM3 cells showing that USP53 overexpression resulted in the deubiquitination of CYCS. IP assays were conducted 48 h after all the plasmids co-transfected. N IP analysis of the ubiquitination of HA-tagged CYCS in Huh-7 cells transfected with mutant USP53 and Flag-tagged USP53. O Western blotting of CYCS poly-ubiquitination. Cells were starved for 4 h before treated with starvation medium containing MG132 for additional 4 h. Cell lysates were immunoprecipitated by anti-CYCS antibody and immunoblotted with TUBE (high affinity ubiquitin binding peptide) following the same method of WB. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001.