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. 2001 Jul 2;194(1):57–70. doi: 10.1084/jem.194.1.57

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© 2001 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Analysis of p21^ras activity and Akt kinase activity of Nf1 ^+/− and wild-type BMMCs stimulated with SCF. (A) p21^ras activity in Nf1 ^+/− and wild-type BMMCs. GTP-bound p21^ras levels were determined by incubating cell lysates with Raf-1 p21^ras binding domain (RBD) agarose beads, fractionating immunoprecipitations by SDS-PAGE, and probing with anti-p21^ras antibody. Immunoblots, quantitative densitometry for p21^ras-GTP levels, and Western blots for total p21^ras are shown. Data are representative of five independent experiments. (B) Akt kinase activity in Nf1 ^+/− and wild-type BMMCs. After preincubation with 100 nM wortmannin or DMSO control for 15 min, Akt kinase activity was determined from Akt immunoprecipitates and an in vitro kinase assay was performed using histone 2B as a phosphorylation substrate. Autoradiography and quantitative densitometry of the phosphorylation of histone 2B and Western blots for total Akt are shown. Data are representative of four independent experiments. Similar inhibition of Akt kinase was observed in cells preincubated for 15 min in 25 μM of LY294002, a second PI-3K inhibitor (data not shown).

Analysis of p21^ras activity and Akt kinase activity of Nf1 ^+/− and wild-type BMMCs stimulated with SCF. (A) p21^ras activity in Nf1 ^+/− and wild-type BMMCs. GTP-bound p21^ras levels were determined by incubating cell lysates with Raf-1 p21^ras binding domain (RBD) agarose beads, fractionating immunoprecipitations by SDS-PAGE, and probing with anti-p21^ras antibody. Immunoblots, quantitative densitometry for p21^ras-GTP levels, and Western blots for total p21^ras are shown. Data are representative of five independent experiments. (B) Akt kinase activity in Nf1 ^+/− and wild-type BMMCs. After preincubation with 100 nM wortmannin or DMSO control for 15 min, Akt kinase activity was determined from Akt immunoprecipitates and an in vitro kinase assay was performed using histone 2B as a phosphorylation substrate. Autoradiography and quantitative densitometry of the phosphorylation of histone 2B and Western blots for total Akt are shown. Data are representative of four independent experiments. Similar inhibition of Akt kinase was observed in cells preincubated for 15 min in 25 μM of LY294002, a second PI-3K inhibitor (data not shown).