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. 2023 Apr 5;14:1881. doi: 10.1038/s41467-023-37277-3

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© The Author(s) 2023

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 — a Structure model (AlphaFold 2.0¹⁸) of CALR del52. Colors represent the difference in relative fractional uptake (ΔRFU) between CALR del52 alone and CALR del52 in complex with TpoR D1-D4 with mature N-glycans. Regions in red and blue are respectively less and more protected in the CALR del52-TpoR D1-D4 complex compared to CALR del52 alone. The scale from red to blue is proportional to the ΔRFU at 1 h incubation in deuterium between indicated species (mean of n = 3 experiments), with dark red and dark blue corresponding to highest differential. Dark gray represents regions without peptide coverage for the given time point. Sequences of the CALR mutant C-terminus are highlighted. Source data are provided as a Source data file. b Representation of N-terminal truncations of CALR del52 fused to either a FLAG tag or a HaloTag at the C-terminus. c NanoBRET between NanoLuc-TpoR (NL-TpoR) or NanoLuc not fused to any protein (NL-Empty) and CALR del52 P-C or C-domain-HaloTag. Data represent mean ± SD (n = 12 biologically independent samples over 4 independent experiments). Data were analyzed by two-ways ANOVA followed by Sidak multiple comparison test. Source data are provided as a Source data file. d Representative co-immunoprecipitation (from 3 independent experiments) of HA-TpoR with CALR del52-FLAG full length or N-terminal truncations as indicated. Source data are provided as a Source data file. e STAT5 transcriptional activity induced by indicated CALR truncations in presence of TpoR. HEK293T were transiently transfected with vectors coding for human TpoR and CALR del52 truncations along with cDNAs coding for STAT5, JAK2 and SpiLuc Firefly luciferase reporter reflecting STAT5 transcriptional activity and normalized with a control reporter (pRLTK) containing Renilla luciferase. Data represent mean ± SD (n = 9 biologically independent samples over 3 independent experiments). Data were analyzed with two-ways ANOVA followed by Sidak multiple comparison test. Source data are provided as a Source data file.