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. 2021 Feb 2;12:734. doi: 10.1038/s41467-020-20820-x

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© The Author(s) 2021

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. 4 — a Schematic illustration showing the design of the proteomics experiments. Tandem Mass Tag (TMT)-based quantitative mass spectrometry (n = 2) was used in VCaP cells overexpressing Control vector (Control), SPOP-WT, or three different SPOP mutants (SPOP-Y87C, SPOP-F102C, and SPOP-W131G). b Scatter plot comparing transcriptomic and proteomic derived fold changes resulting from the comparison between SPOP-MTs (average across SPOP-Y87C, -F102C, -W131G) and SPOP-WT VCaP cells. (n = 3 for transcriptome, n = 2 for proteome). Genes belonging to DHT-Induced/ERG-Repressed gene-signature (AR + ERG co-bound) are highlighted in red. TRIM24 and ZMYND11 are the most upregulated proteins without changes at mRNA levels and are highlighted in green. CDKN1A (p21) upregulated at both mRNA and protein levels, and MYC downregulated at both mRNA and protein levels are highlighted in black. c Over-expression of HA-ZMYND11 and SPOP-WT in 293T cells and subsequent expression analysis of the indicated proteins by immunoblotting (n = 2). d Whole-cell extracts (WCE) of 293T cells over-expressing HA-ZMYND11-WT and different SPOP species and corresponding anti-HA-immunoprecipitation (HA-IP). Expression of the indicated proteins was analyzed by immunoblotting (n = 1). e Domain structure of ZMYND11 with indicated SPOP-degron and ubiquitin sites. f Forced expression of SPOP-WT together with HA-ZMYND11-WT or two degron-deficient mutants (DMT1 & DMT2) in 293T cells (n = 1). g In vivo ubiquitylation assay of HA-ZMYND11 in 293T cells. Cells were transiently transfected with the indicated constructs, and histidine-tagged (his-tag), ubiquitylated proteins were pulled down using nickel beads. Ubiquitylated HA-tagged ZMYND11 was detected by immunoblotting (n = 1). h Over-expression of HA-ZMYND11 and SPOP-Y87C in 293T cells and subsequent expression analysis of the indicated proteins by immunoblotting after proteasomal inhibition with MG132. i Whole-cell extracts (WCE) and corresponding anti-HA-immunoprecipitation (HA-IP) of 293T cells over-expressing HA-ZMYND11-WT and different SPOP-MTs species as indicated. Expression of the indicated proteins was analyzed by immunoblotting (n = 1). j In vivo ubiquitylation assay of HA-ZMYND11 in 293T cells. Cells were transiently transfected with the indicated constructs and histidine-tagged (His-tag); ubiquitylated proteins were pulled down using nickel beads. Ubiquitylated HA-tagged ZMYND11 was detected by immunoblotting (n = 1). k Immunoblots of indicated proteins in VCaP, LNCaP, and LAPC4 human prostate cancer cells overexpressing the indicated SPOP species. Molecular weights are indicated in kilodaltons (kDa) (n = 3). Source data are provided as a Source Data File.