Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2019 Mar 28;23:101175. doi: 10.1016/j.redox.2019.101175

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2019 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 6 — Covalent modification of STAT3 by15-keto PGE₂. A. A proposed interaction between 15-keto PGE₂ and a thiol group of STAT3. B. MCF10A-ras cells were pretreated with a thiol reducing agent, DTT (100 μM), for 1 h followed by exposure to 15-keto PGE₂ (20 μM) for an additional 24 h. The protein levels of P-STAT3^Y705 and STAT3 were determined by Western blot analysis. **p < 0.01, ***p < 0.001. C. The direct binding of 15-keto PGE₂ to STAT3 was assessed using the avidin-biotin system. MCF10A-ras cells were pretreated with DTT (100 μM) for 1 h and then treated with biotinylated 15-keto PGE₂ (40 μM) for an additional 12 h. The biotinylated 15-keto PGE₂-STAT3 complex was detected by the immunoprecipitation and Western blot analyses. D. A putative model of 15-keto PGE₂ covalently bound to Cys259 or Cys251 of STAT3 as predicted by a docking study. The blue dotted line represents a H-bond. E. PC3 cells were transfected with WT or Cys251 or Cys259-mutated STAT3 followed by treatment with 15-keto PGE₂ (40 μM) for 24 h. WT STAT3 and STAT3 mutants were detected by anti-STAT3 antibody. F. Annotated fragment MS/MS spectrum of 15-keto PGE₂ in positive ESI mode. The elucidation of fragment ion at m/z 333.2073, 315.1951 from the structure is given in the inset of the spectra. G. Mass spectrum of the STAT3 peptide (RQQIACIGGPPNICLDR) with 15-keto PGE₂ binding. Annotated MS/MS spectrum illustrating the binding of 15-keto PGE₂ to STAT3 protein at cysteine 259 residues was identified by LC-MS/MS. An exclamation mark indicates a carbamidomethylation on Cys251, and an asterisk indicates 15-keto PGE₂ binding to Cys259. The precursor ion [M+3H]³ ⁺ for peptide with 15-keto PGE₂ binding is m/z 754.0403. The 15-keto PGE₂ fragment ions are inserted into the spectrum as a table. Each molecular formula present with b and y ions on the spectrum represents the 15-keto PGE₂ fragment ion bound on Cys259. The sample preparation and other experimental details for mass spectral analysis are described in the Materials and methods. Sequence-informative fragmentation ions are summarized on the peptide sequence.

Fig. 6 — Covalent modification of STAT3 by15-keto PGE₂. A. A proposed interaction between 15-keto PGE₂ and a thiol group of STAT3. B. MCF10A-ras cells were pretreated with a thiol reducing agent, DTT (100 μM), for 1 h followed by exposure to 15-keto PGE₂ (20 μM) for an additional 24 h. The protein levels of P-STAT3^Y705 and STAT3 were determined by Western blot analysis. **p < 0.01, ***p < 0.001. C. The direct binding of 15-keto PGE₂ to STAT3 was assessed using the avidin-biotin system. MCF10A-ras cells were pretreated with DTT (100 μM) for 1 h and then treated with biotinylated 15-keto PGE₂ (40 μM) for an additional 12 h. The biotinylated 15-keto PGE₂-STAT3 complex was detected by the immunoprecipitation and Western blot analyses. D. A putative model of 15-keto PGE₂ covalently bound to Cys259 or Cys251 of STAT3 as predicted by a docking study. The blue dotted line represents a H-bond. E. PC3 cells were transfected with WT or Cys251 or Cys259-mutated STAT3 followed by treatment with 15-keto PGE₂ (40 μM) for 24 h. WT STAT3 and STAT3 mutants were detected by anti-STAT3 antibody. F. Annotated fragment MS/MS spectrum of 15-keto PGE₂ in positive ESI mode. The elucidation of fragment ion at m/z 333.2073, 315.1951 from the structure is given in the inset of the spectra. G. Mass spectrum of the STAT3 peptide (RQQIACIGGPPNICLDR) with 15-keto PGE₂ binding. Annotated MS/MS spectrum illustrating the binding of 15-keto PGE₂ to STAT3 protein at cysteine 259 residues was identified by LC-MS/MS. An exclamation mark indicates a carbamidomethylation on Cys251, and an asterisk indicates 15-keto PGE₂ binding to Cys259. The precursor ion [M+3H]³ ⁺ for peptide with 15-keto PGE₂ binding is m/z 754.0403. The 15-keto PGE₂ fragment ions are inserted into the spectrum as a table. Each molecular formula present with b and y ions on the spectrum represents the 15-keto PGE₂ fragment ion bound on Cys259. The sample preparation and other experimental details for mass spectral analysis are described in the Materials and methods. Sequence-informative fragmentation ions are summarized on the peptide sequence.