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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. 4 — a A schematic diagram representing the PrLZ structural domains for mapping the interaction domains with SPOP. b Immunoblot (IB) analysis of WCL and anti-HA immunoprecipitates (IPs) derived from 293 T cells transfected with GST-SPOP and indicated constructs of HA-PrLZ. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. WT, wild type. c IB analysis of WCL derived from 293 T cells transfected with HA-PrLZ WT, HA-PrLZ animo acid (aa) 46-224 constructs and increasing transfection doses (1 and 3 μg) of Flag-SPOP. WT, wild type. d IB analysis of WCL and anti-Flag IPs derived from 293 T cells transfected with HA-SPOP, Flag-PrLZ WT, and deletion of aa 30-42 domain-PrLZ constructs. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. WT, wild type. e IB analysis of WCL derived from 293 T cells transfected with HA-SPOP, Flag-PrLZ WT, and deletion of aa 30-42 domain-PrLZ constructs. EV, empty vector. WT, wild type. f IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with HA-SPOP, His-Ub, Flag-PrLZ WT, and deletion of aa 30-42 domain-PrLZ constructs. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. EV, empty vector. WT, wild type. g IB analysis of WCL and anti-Flag IPs derived from 293 T cells transfected with HA-SPOP and indicated mutation constructs of Flag-PrLZ. EV, empty vector. WT, wild type. h IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with HA-SPOP, His-Ub, Flag-PrLZ WT, and Flag-PrLZ S40A mutant. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. EV, empty vector. WT, wild type. i IB analysis of WCL derived from 293 T cells transfected with HA-SPOP, Flag-PrLZ WT, and Flag-PrLZ S40A mutant. Where indicated, 100 μg/ml CHX was added for the indicated time period before harvesting. WT, wild type. j The PrLZ protein abundance in (i) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. k IB analysis of WCL derived from 293 T cells transfected with HA-SPOP and pull-down binding assay using biotinylated peptide.