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

. 2015 Sep 30;5:14538. doi: 10.1038/srep14538

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

Copyright © 2015, Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

PMC Copyright notice

(a,b) Microscale thermophoresis (MST) analysis showing the dual binding of fedratinib to JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Fedratinib binding was carried out with label free proteins. (c,d) MST analysis shows a single binding site (Site I-ATP site) for ruxolitinib with JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Because, ruxolitinib showed auto-fluorescence that precluded label free binding analysis, we performed ruxolitinib binding with fluorescently labeled proteins. Note, we could not detect any secondary binding site for ruxolitinib. (e,f) MST analysis with JAK2-L983F showed only one binding site for fedratinib at site –II (substrate site). A phenylalanine substitution for Leu 983 causes steric hindrance to fedratinib and it no longer can bind to site-I, ATP site. In contrast, substrate site is not affected by this mutation and therefore binding of fedratinib to site-II is not affected showing similar kd values. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. (g) MST analysis with JAK2-L983F showing loss of ruxolitinib binding with mutant L983F. These experiments were performed twice in duplicates.