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. 2022 Feb 22;29(8):1611–1624. doi: 10.1038/s41418-022-00951-y

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© The Author(s) 2022

Open AccessThis 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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Fig. 2 — a PrLZ binds with SPOP. Immunoblot (IB) analysis of WCL and anti-HA immunoprecipitates (IPs) derived from 293 T cells transfected with HA-PrLZ and Flag-tagged BTB domain-containing protein constructs. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. b IB analysis of WCL and anti-SPOP IPs derived from C4-2. Cells were treated with 20 μM MG132 for 6 h before harvesting. c GST pull-down assay revealed the direct interaction between PrLZ and SPOP. The upper panel presents the result of IB by using the antibody against His, and the lower coomassie blue staining showing the gels for purified proteins. d IB analysis of WCL derived from C4-2 cells with SPOP knockout by the CRISPR-Cas9 technology. Parental C4-2 cells are used as the control. e IB analysis of WCL derived from 22Rv1 cells with SPOP knockout by the CRISPR-Cas9 technology. Parental 22Rv1 cells are used as the control. f IB analysis of WCL derived from C4-2 cells transfected with increasing transfection doses (1 and 2 μg) of HA-SPOP. EV, empty vector. g SPOP knockout cells (sgSPOP), as well as parental C4-2 cells (Control), were treated with 100 μg/ml CHX for the indicated time period before harvesting. Equal amounts of WCL were immunoblotted with the indicated antibodies. h The PrLZ protein abundance in (g) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. i IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP, Myc-Cullin 3 and His-Ub. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. j IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP and K-only ubiquitin mutants. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. k IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP and the indicated ubiquitin KR (Lys to Arg) mutants. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. l IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-tagged wild type (WT) and mutanted PrLZ, HA-SPOP, and His-Ub. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. m The growth curve of C4-2 cells with knockdown of SPOP and/or PrLZ. Scr, Scramble. *P < 0.05. n, o Colony formation assays and quantification of C4-2 cells with knockdown of SPOP and/or PrLZ. Scr, Scramble. Error bars represent SEs. *P < 0.05. p, q Representative images and quantification of migrated C4-2 cells with knockdown of SPOP and/or PrLZ. Scr, Scramble. Error bars represent SEs. *P < 0.05.