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. 2018 Nov 16;9:4700. doi: 10.1038/s41467-018-07178-x

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© The Author(s) 2018

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. 2 — STUB1/HSP70 complex regulates AR variants protein expression. a Core-enrichment genes of ubiquitin-mediated proteolysis pathway in C4-2B parental, C4-2B MDVR, and C4-2B AbiR cells. The red arrow showed STUB1 was suppressed in resistant cells. b mRNA levels of STUB1 in LNCaP, C4-2B parental, C4-2B AbiR, C4-2B MDVR, CWR22Rv1, and VCaP cells was examined by real-time PCR (left). Protein expression of STUB1, AR-V7, AR-FL was determined by western blot (right). c 293 cells were transiently transfected with Flag-STUB1 or HSP70 with or without vector, AR-V1, AR-V3, AR-V7, AR-V9, or AR-V12 for 3 days and whole cell lysates were immunopreciptated with anti-Flag or HSP70 antibodies and bloted with indicated antibodies. d CWR22Rv1 and C4-2B MDVR cells were transiently transfected with 2 or 5 µg Flag-STUB1 for 3 days, whole cell lysates were collected and subjected to western blot. e CWR22Rv1 cells were transiently transfected with vector or flag-STUB1 and then treated with 50 µg/mL cycloheximide, total cell lysates were collected at 0, 2, 4, and 8 h after the treatment and subjected to western blot. Half-lives of AR-V7 and AR-FL were calculated. f 293 cells were transiently transfected with AR-V7 with or without STUB1 for 3 days and then treated with 5 µM MG132 overnight. Total cell lysates were collected and subjected to western blot. g 293 cells were transiently transfected with AR-V7, flag-STUB1, and HA-ubiquitin plasmids for 3 days, following treatment with or without 5 µM MG132 for additional 6 h. Total cell lysates were collected and immunopreciptated with AR-V7 antibody and blotted with indicated antibodies. h 293 cells were co-transfected with HSP70, AR-V7 with or without Flag-STUB1 for 3 days and then whole cell lysates were immunopreciptated with anti-HSP70 antibody and blotted with indicated antibodies. i C4-2B MDVR or CWR22Rv1 cells were transiently transfected with STUB1 and then treated with 20 μM enzalutamide or 5 μM abiraterone for 3 days and total cell number was determined. *p < 0.05. Results are the mean of three independent experiments (±s.d.). Statistical analysis was performed using one-way ANOVA. AR-FL: full-length AR, AR-Vs: AR-Variants, Ub: Ubiquitin