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. 2021 Jan 4;4:36. doi: 10.1038/s42003-020-01528-6

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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. 1 — A Schematic of the peptide-capture technique used to identify analog-sensitive (AS) PIM1 substrates and phosphorylation sites in prostate cancer cells. AS PIM1 uses ATPγS, a bulky ATP analog, to thiophosphorylate substrates. Upper panel: thiophosphorylated substrates are alkylated by p-nitrobenzyl mesylate (PNBM) and recognized by an antibody to the thiophosphate ester moiety (ThioP). Lower panel: thiophosphorylated peptides are captured on a resin, eluted, and identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). B ATP analog optimization in LNCaP cells stably expressing either WT PIM1 or AS PIM1. Cells were treated with indicated ATP analog (ATP = ATPγS; PheET = N⁶-Phenylethyl ATPγS) in the presence of digitonin, a mild-permeabilizing agent. Alkylation was completed using PNBM. Whole-cell lysates were analyzed by western blot for the presence of thiophosphorylation (ThioP), PIM1 (PIM1), and HSP90 as loading control. C AS PIM1 thiophosphorylates endogenous AR in LNCaP cells. LNCaP-WT PIM1 and LNCaP-AS PIM1 cells were treated PhET-ATPγS as above. AR was immunoprecipitated and analyzed by western blot for the presence of thiophosphorylation (thioP) and AR. Input shows the protein levels in cells of PIM1, as well as the abundance of immunoprecipitated AR. Tubulin acts as a loading control. Western blots are representative of two independent experiments.