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. 2022 Aug 15;13:4788. doi: 10.1038/s41467-022-32541-4

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© The Author(s) 2022

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. 4 — a DLC1 RhoGAP wild-type (WT, blue and cyan) but not R1114A mutant (red and pink) stimulates RhoA GTPase activity. The concentration of phosphate generated (μM) with added GTP (+GTP) or without GTP (−GTP) is shown. Data are shown as mean values (bars) +/− SD (error bars), and individual measurements are plotted (dots, n = 2). b Titration of p120RasGAP SH3 domain (0–20 μM) wild type (WT, blue) and structurally defined mutants Y290A (red) and E298A (green) to inhibit DLC1 RhoGAP activity. The phosphate generated (in μM) is plotted against the log10 of SH3 concentration (μM), IC₅₀ values were calculated by nonlinear regression (lines), and individual measurements are plotted (dots, n = 4). c Representative SH3 domain concentrations from (b) (2, 3, 6, and 10 μM). % GAP activity is normalized to the maximum phosphate generated by DLC1 stimulated RhoA (black bar, 100%). Data are mean values (bar graph) +/− SD (error bars), and individual measurements are plotted (dots, n = 4). P values: WT vs Y290A: 2 μM SH3: P = 0.0414; 3 μM SH3: P = 0.0263; 6 μM SH3: P = 0.0070; 11 μM SH3: P = 0.0346; WT vs E298A: 2 μM SH3: P = 0.0455; 3 μM SH3: P = 0.0041; 6 μM SH3: P = 0.0004; 11 μM SH3: P = 0.0004. d Inhibition of DLC1 RhoGAP activity by p120RasGAP SH2-SH3-SH2 wild type (blue) or E298A mutant (red), and SH3 domain (green). The phosphate signal (μM) is plotted against the log10 of p120RasGAP concentration. IC₅₀ values were calculated by nonlinear regression (lines), and individual measurements are plotted (dots, n = 7). In b, d, IC₅₀ values are calculated by nonlinear regression in GraphPad Prism using the One site–fit logIC50 model, and are reported in Table 2. In c, P values are calculated in GraphPad Prism using ordinary one-way ANOVA analysis with Tukey’s multiple comparison test. Significant differences are based on P values as indicated:* P = 0.01 to 0.05;** P = 0.001 to 0.01;*** P = 0.0001 to 0.001. Source data are available as a Source Data file.