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. 2019 Dec 20;10:5828. doi: 10.1038/s41467-019-13782-2

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© The Author(s) 2019

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. 7 — a The kinase activity of endogenous c-Abl is elevated after hypo-osmotic treatment. Endogenous c-Abl was immunoprecipitated from cells treated with iso-osmotic or hypo-osmotic medium (60 mOsm, 10 min) and in vitro kinase assay was performed using GST-CrkII as substrate. Phosphorylation (radiolabeled) and the total protein (Coomassie stained) signals are shown. N = 3 (control) and n = 5 (hypo-osmotic) biologically independent samples from 3 independent experiments. Statistical analysis with a two-tailed unpaired t test. *P < 0.05. b Kinetics of c-Abl activation upon osmotic swelling (30 mOsm) and after shock release is shown. Endogenous CrkII was immunoprecipitated from cells and pY221CrkII and CrkII was detected, normalized to iso-osmotic. From left to right, n = 5, 3, 5, 9, 2, 2, from biologically independent samples from the corresponding independent experiments. Statistical analysis with a two-tailed unpaired t test. *P < 0.05. c c-Abl kinase mechanosenses substrate rigidity. Endogenous CrkII was immunoprecipitated from cells grown in soft or stiff hydrogels and pY221CrkII and CrkII was detected. N = 4 biologically independent samples from 4 independent experiments. Statistical analysis with a two-tailed unpaired t test. *P < 0.05. d c-Abl kinase is activated by mechanical strain. Endogenous CrkII was immunoprecipitated from human fibroblasts unstretched and stretched for 10 min and pY221CrkII and CrkII was detected. ^#P = 0.08, n = 9 from biologically independent samples from 5 independent experiments. e Cartoon representing the different tensional states of a cell in culture and the activity of c-Abl depending on those tensional states. f Schematic representation of the heteropolyprotein (I91 ΔCys)₂-ABD-(I91 ΔCys)₂ used to characterize the mechanical properties of ABD. g Typical force-extension trace showing four unfolding peaks of the fingerprinting domain I91, which are preceded by a featureless extension which suggests that ABD unfolds at low forces. Solid lines show worm-like chain plots used to estimate contour lengths. h Final contour lengths of fingerprinted single-molecule force-extension traces. Dashed lines indicate theoretical final contour length values if ABD unfolds during the experiment (black) or remains folded (gray) are indicated. Data represent mean ± S.E.M.