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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. 3 — STUB1/HSP70 complex regulates sensitivity to anti-androgens. a Total mRNA and whole cell lysates from LNCaP, C4-2B, C4-2B AbiR, C4-2B MDVR, and CWR22Rv1 cells were collected and subjected to qRT-PCR and western blot, respectively. b 293 cells were co-transfected with Flag-STUB1, AR-V7, HA-Ub with or without HSP70 for 3 days, whole cell lysates were immunopreciptated with anti-Flag or AR-V7 antibodies and blotted with AR-V7, HSP70, Flag or HA antibodies. c CWR22Rv1 and C4-2B MDVR cells were transiently transfected with two independent HSP70 siRNA (#5 and #6) and then treated with 20 μM enzalutamide. Total cell number was determined at 3 and 6 days. d C4-2B MDVR and CWR22Rv1 cells were transiently transfected with two independent HSP70 siRNA, whole cell lysates were collected and subjected to western blot. PSA luciferase activity was determined. e IMR90, PZ-HPV7, C4-2B MDVR, and CWR22Rv1 cells were treated with different concentrations of APO and VER for 3 days, total cell numbers were determined. f C4-2B MDVR and CWR22Rv1 cells were treated with 5 and 10 μM APO or VER for 48 h, total cell lysates were collected and subjected to western blot. g C4-2B MDVR cells were treated with 5 μM APO and VER with or without 20 μM enzalutamide or 5 μM abiraterone, total cell number was determined at 3 and 6 days. h C4-2B MDVR cells were treated with 5 μM APO or VER with or without enzalutamide and abiraterone, colonogenic assay was performed and colonies were quantified. *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, ENZA: Enzalutamide, AA: Abiraterone acetate, APO: Apoptozole, VER: Ver155008, RLU: Relative luciferase unit