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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. 1 — a Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from C4-2 cells treated with MG132 (5 and 10 μM) or MLN4924 (0.5 and 1 μM) for 12 h. b IB analysis of WCL derived from 22Rv1 cells under similar treatment condition. c PrLZ binds with Cullin 1 and Cullin 3. IB analysis of WCL and anti-Myc immunoprecipitates (IPs) derived from 293 T cells transfected with Flag-PrLZ and indicated Myc-tagged Cullins. EV, empty vector. d IB analysis of WCL and anti-Flag IPs derived from 293 T cells transfected with Flag-PrLZ and Myc-Cullin 3. EV, empty vector. e IB analysis of WCL derived from C4-2 and 22Rv1 cells stably expressing shCullin 3 or shScr. Scr, Scramble. f IB analysis of WCL derived from C4-2 cells transfected with increasing transfection doses (0.5, 1.5 and 3 μg) of Myc-Cullin 3. EV, empty vector. g IB analysis of WCL derived from 293 T cells transfected with Flag-PrLZ and increasing transfection doses (1.5 and 3 μg) of Myc-Cullin 3. EV, empty vector. h IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, Myc-Cullin 3, and His-Ub. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. i Cullin 3 knockdown cells (shCullin 3), as well as parental C4-2 cells (shScr), were treated with 100 μg/ml cycloheximide (CHX) for the indicated time period before harvesting. Equal amounts of WCL were immunoblotted with the indicated antibodies. j The PrLZ protein abundance in (i) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin.