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Results: 5

1.
Fig. 5

Fig. 5. From: A Ceramide-binding C1 Domain Mediates Kinase Suppressor of Ras Membrane Translocation.

FB1 inhibits KSR1 membrane translocation and kinase activation. COS-7 cells were treated with increasing doses of FB1 for 16 h and ceramide levels were measured using the diacylglycerol kinase assay as described in Materials and Methods (A). In (B), COS-7 cells transfected with Myc-KSR1 were pretreated with or without 100 µM FB1 for 16 h and then stimulated with increasing doses of EGF. Myc-KSR1 membrane translocation was determined by indirect immunofluorescence staining and quantified using fluorescence microscopy. In (C), Myc-KSR1 kinase activity was analyzed the using two-stage in vitro reconstitution assay. Results represent one of three similar experiments in (A-C).

Xianglei Yin, et al. Cell Physiol Biochem. 2009 August;24(3-4):219-230.
2.
Fig. 1

Fig. 1. From: A Ceramide-binding C1 Domain Mediates Kinase Suppressor of Ras Membrane Translocation.

CA3 domain mutant CA3 (C359S/C362S) is defective in ceramide binding. An ELISA was used to measure the binding of the KSR1 CA3 domain to ceramide. (A) The wells of the Nunc-MaxiSorp plate were coated with 8 nmol/well BSA-conjugated C16-ceramide (CER) or BSA-conjugated C16-dihydro-ceramide (DH-CER). The binding of anti-ceramide Ab with ceramide (left panel) was detected by sequential incubation with HRP-conjugated anti-mouse IgM secondary Ab and TMB substrate solution as in Materials and Methods; the binding of recombinant GST-CA3, GST-CA3 (CS) and GST proteins with ceramide (right panel) was detected by sequential incubation with HRP-conjugated anti-GST Ab and TMB substrate solution. In (B), microtiter wells were coated with 8 nmol/well non-BSA-conjugated C16-ceramide and DAG or, in (C) with different ceramide species, GM1, SM, PC and DAG. The binding of recombinant GST-CA3, GST-CA3 (CS) and GST proteins with ceramide and other lipids was detected as in (A, right panel). Data are representative of one of three independent experiments in (A-C).

Xianglei Yin, et al. Cell Physiol Biochem. 2009 August;24(3-4):219-230.
3.
Fig. 4

Fig. 4. From: A Ceramide-binding C1 Domain Mediates Kinase Suppressor of Ras Membrane Translocation.

KSR1 antisense ODN impairs EGF-induced c-Raf-1 translocation into sphingolipid-rich light membranes. MCF-7 cells, transfected with 6 µM KSR1 AS-ODN or sense-ODN, were either left untreated or treated with 10 ng/ml EGF for 3 min. The expression level of endogenous KSR1 and the phosphorylation of MEK1/2 were determined by Western blot analysis using anti-KSR1 and anti-phospho-MEK1/2 Abs as described in Materials and Methods. Equal loading of samples were confirmed using β-actin (A, upper panel). Proteins were also subjected to sucrose gradient fractionation as above, and the presence of c-Raf-1 in the light membrane fraction was visualized by Western blot using anti-c-Raf Ab. Flotillin-2 was used as a raft marker (A, lower panel). In EGF-stimulated MCF-7 cells, down-regulation of KSR1 expression and the attenuated MEK1/2 phosphorylation and c-Raf-1 recruitment into the raft fraction after AS-ODN treatment were quantified by densitometry and presented as arbitrary optical density units (B). These results represent one of three similar experiments.

Xianglei Yin, et al. Cell Physiol Biochem. 2009 August;24(3-4):219-230.
4.
Fig. 2

Fig. 2. From: A Ceramide-binding C1 Domain Mediates Kinase Suppressor of Ras Membrane Translocation.

An intact CA3 domain of KSR1 is required for its translocation to plasma membrane and kinase activation upon EGF stimulation, and for its enhancement of COS-7 cell proliferation. COS-7 cells transiently transfected with Myc-KSR1, Myc-Ki-KSR1 or Myc-KSR1-CS were serum starved for 12 h before treatment with 10 ng/ml EGF for the indicated times or increasing EGF doses for 3 min. Membrane translocation of KSR1 was determined by indirect immunofluorescence staining with anti-Myc Ab and quantified using fluorescence microscopy. Representative images are shown in (A). Data (mean±SD) are collated from three independent experiments in which 200 cells were analyzed per point (B). In (C), kinase activity of Myc-KSR1 or Myc-KSR1-CS mutant, treated 12 h post serum-starvation with 10 ng/ml EGF for the indicated times or with increasing dose of EGF for 3 min, was measured using the two-stage in vitro reconstitution assay as described in Materials and Methods. Results represent one of three independent experiments. In (D), proliferation of COS-7 cells stably-expressing wild type Myc-KSR1 or mutant Myc-KSR1-CS was monitored using WST-1 reagent. The insert shows the expression level of Myc-KSR1 and Myc-KSR1-CS in the stably-transfected cell lines. Data (mean±SD) are compiled from three independent experiments.

Xianglei Yin, et al. Cell Physiol Biochem. 2009 August;24(3-4):219-230.
5.
Fig. 3

Fig. 3. From: A Ceramide-binding C1 Domain Mediates Kinase Suppressor of Ras Membrane Translocation.

KSR1 is recruited into a ceramide-rich surface macro-domain and activated upon EGF stimulation. COS-7 cells, transiently transfected with Myc-KSR1, either left untreated or stimulated with 10 ng/ml EGF for 3 min, were double-stained for KSR1 and GM1 with anti-Myc Ab and FITC-conjugated cholera toxin subunit B (CTXB), respectively (A); for KSR1 and caveolin-1 with anti-Myc Ab and anti-caveolin-1 Ab, respectively (B); or for KSR1 and ceramide with anti-Myc Ab and anti-ceramide Ab, respectively (C). Immunofluorescent staining was analyzed by confocal microscopy. KSR1 co-localization with GM1, caveolin-1 or ceramide at the plasma membrane after EGF stimulation is identified as cell surface yellow staining in overlay images. Representative images from one of three similar experiments are shown. In (D), cell lysates from Myc-KSR1 or Myc-KSR1-CS transfected COS-7 cells, treated with or without 10 ng/ml EGF, were separated using discontinuous sucrose gradient fractionation. Protein in each fraction was TCA precipitated, separated by SDS-PAGE and immuoblotted with antibodies against Flotillin-1 or Myc. The sucrose and total protein profiles after centrifugation are shown below. These results represent one of three independent experiments. In (E), Myc-KSR1 was immunopre-cipitated from Light Fraction (LF, combined fractions 4-9), Heavy Fraction (HF, combined fractions 11 and 12) and Pellet (P), prepared as described above, and kinase activity was measured using the two-stage in vitro reconstitution assay. Arbitrary KSR1 kinase activity was quantified by densitometry and normalized to levels of immunopre-cipitated KSR1. These results represent one of three independent experiments.

Xianglei Yin, et al. Cell Physiol Biochem. 2009 August;24(3-4):219-230.

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