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Results: 12

1.
Figure 5

Figure 5. From: Signal Transduction by Cxc Chemokine Receptor 4.

Downregulation of CXCR4 by SDF-1. PBMCs were incubated in RPMI supplemented with 10% FCS without (control, 100% expression) or with increasing concentrations of SDF-1 for the times indicated. Surface-expressed CXCR4 was detected with mAb 6H8 stained with PE-conjugated goat anti–mouse IgG using a FACScan™ flow cytometer. The percentage of CXCR4 surface expression was calculated from the relative fluorescence intensity (see Materials and Methods). The results shown are representative for four experiments obtained with cells from different donors.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
2.
Figure 7

Figure 7. From: Signal Transduction by Cxc Chemokine Receptor 4.

Inhibition of CD26/DPPIV (dipeptidyl dipeptidase IV) prolongs SDF-1 stimulated protein kinase B activation. T cells were incubated with 30 nM SDF-1 in the absence (Control) or presence of 4 mM of the CD26/DPPIV inhibitor diprotin A for the indicated times. Activated (pSer473PKB) and total protein kinase B (PKB) were determined by Western blot analysis as in the legend to Fig. 1 A.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
3.
Figure 2

Figure 2. From: Signal Transduction by Cxc Chemokine Receptor 4.

Effect of the CXCR4 antagonist, SDF-1P2G, on SDF-1–induced protein kinase B activation. (A) T cells were pretreated with 10 μM SDF-1P2G (P2G) or medium for 5 min and stimulated with 100 nM SDF-1 for 10 min. (B) The cells were stimulated with 100 nM SDF-1 for 5, 10, or 15 min. Where indicated, 10 μM SDF-1P2G (P2G) was added 5 min before termination of the stimulation. Activated (pSer47 3PKB) and total protein kinase B(PKB) were determined as in the legend to Fig. 1 A. (C) Protein kinase B activity in immunoprecipitates. The cells were either treated with medium (Cont), 100 nM SDF-1 (SDF-1), or 10 μM SDF-1P2G (P2G) for 10 min, or treated with 10 μM SDF-1P2G for 5 min before stimulation with 100 nM SDF-1 for 10 min (P2G > SDF-1), or were stimulated with 100 nM SDF-1 and 5 min later treated with 10 μM SDF-1P2G for an additional 5 min (SDF-1 > P2G). Results are expressed as in Fig. 1 C. Similar results were obtained with cells from three different donors.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
4.
Figure 2

Figure 2. From: Signal Transduction by Cxc Chemokine Receptor 4.

Effect of the CXCR4 antagonist, SDF-1P2G, on SDF-1–induced protein kinase B activation. (A) T cells were pretreated with 10 μM SDF-1P2G (P2G) or medium for 5 min and stimulated with 100 nM SDF-1 for 10 min. (B) The cells were stimulated with 100 nM SDF-1 for 5, 10, or 15 min. Where indicated, 10 μM SDF-1P2G (P2G) was added 5 min before termination of the stimulation. Activated (pSer47 3PKB) and total protein kinase B(PKB) were determined as in the legend to Fig. 1 A. (C) Protein kinase B activity in immunoprecipitates. The cells were either treated with medium (Cont), 100 nM SDF-1 (SDF-1), or 10 μM SDF-1P2G (P2G) for 10 min, or treated with 10 μM SDF-1P2G for 5 min before stimulation with 100 nM SDF-1 for 10 min (P2G > SDF-1), or were stimulated with 100 nM SDF-1 and 5 min later treated with 10 μM SDF-1P2G for an additional 5 min (SDF-1 > P2G). Results are expressed as in Fig. 1 C. Similar results were obtained with cells from three different donors.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
5.
Figure 4

Figure 4. From: Signal Transduction by Cxc Chemokine Receptor 4.

SDF-1 concentration dependence of prolonged protein kinase B and ERK-2 activation. (A) Western blot analysis of protein kinase B. T cells were stimulated with 10, 30, 100 nM or 1 μM SDF-1 for the indicated times. Activated (pSer473 PKB) and total protein kinase B (PKB) were determined as in the legend to Fig. 1 A. (B) Protein kinase B activity in immunoprecipitates from lysates of cells stimulated with the indicated concentrations of SDF-1 for either 1 min (•) or 20 min (○). Results are expressed as fold activation. (C) Western blot analysis of ERK-2. T cells were stimulated with 10, 100 nM and 1 μM SDF-1 for the indicated times. Activated ERK-2 (pERK-2) corresponds to the band with retarded electrophoretic mobility. Results are representative for at least three independent determinations.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
6.
Figure 3

Figure 3. From: Signal Transduction by Cxc Chemokine Receptor 4.

Effect of wortmannin on SDF-1–induced protein kinase B activation and PIP3 production. (A) T cells were pretreated with 100 nM wortmannin for 10 min and stimulated with 100 nM SDF-1 for 10 min. (B) The cells were stimulated with 100 nM SDF-1 for 1, 5, 10, and 15 min. Where indicated, 100 nM wortmannin was added 5 min before termination of the stimulation. Activated (pSer473PKB) and total protein kinase B (PKB) were determined as in the legend to Fig. 1 A. (C) Protein kinase B activity in immunoprecipitates. The cells were either treated with medium (Cont), 100 nM SDF-1 (SDF-1), or 100 nM wortmannin (wort) for 10 min, or were treated with 100 nM wortmannin for 10 min before stimulation with 100 nM SDF-1 for 10 min (wort > SDF-1), or were stimulated with 100 nM SDF-1 and 5 min later treated with 100 nM wortmannin for an additional 5 min (SDF-1 > wort). Results are expressed as in Fig. 1 C. D. PIP3 formation in [32P]orthophosphate-loaded T cells. The cells were either treated with medium (Cont), 100 nM SDF-1 (SDF-1 10′), 100 nM wortmannin (wort), 10 µM SDF-1P2G (P2G) for 10 min or with 100 nM SDF-1 for 1 min (SDF-1 1′), or were stimulated with 100 nM SDF-1 and 5 min later treated with 100 nM wortmannin (SDF-1 > wort) or 10 μM SDF-1P2G (SDF-1 > P2G) for an additional 5 min. Lipids were extracted, deacylated, and glycerophosphoinositides were separated by HPLC (duplicate determinations). Results are representative for at least three independent experiments.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
7.
Figure 3

Figure 3. From: Signal Transduction by Cxc Chemokine Receptor 4.

Effect of wortmannin on SDF-1–induced protein kinase B activation and PIP3 production. (A) T cells were pretreated with 100 nM wortmannin for 10 min and stimulated with 100 nM SDF-1 for 10 min. (B) The cells were stimulated with 100 nM SDF-1 for 1, 5, 10, and 15 min. Where indicated, 100 nM wortmannin was added 5 min before termination of the stimulation. Activated (pSer473PKB) and total protein kinase B (PKB) were determined as in the legend to Fig. 1 A. (C) Protein kinase B activity in immunoprecipitates. The cells were either treated with medium (Cont), 100 nM SDF-1 (SDF-1), or 100 nM wortmannin (wort) for 10 min, or were treated with 100 nM wortmannin for 10 min before stimulation with 100 nM SDF-1 for 10 min (wort > SDF-1), or were stimulated with 100 nM SDF-1 and 5 min later treated with 100 nM wortmannin for an additional 5 min (SDF-1 > wort). Results are expressed as in Fig. 1 C. D. PIP3 formation in [32P]orthophosphate-loaded T cells. The cells were either treated with medium (Cont), 100 nM SDF-1 (SDF-1 10′), 100 nM wortmannin (wort), 10 µM SDF-1P2G (P2G) for 10 min or with 100 nM SDF-1 for 1 min (SDF-1 1′), or were stimulated with 100 nM SDF-1 and 5 min later treated with 100 nM wortmannin (SDF-1 > wort) or 10 μM SDF-1P2G (SDF-1 > P2G) for an additional 5 min. Lipids were extracted, deacylated, and glycerophosphoinositides were separated by HPLC (duplicate determinations). Results are representative for at least three independent experiments.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
8.
Figure 3

Figure 3. From: Signal Transduction by Cxc Chemokine Receptor 4.

Effect of wortmannin on SDF-1–induced protein kinase B activation and PIP3 production. (A) T cells were pretreated with 100 nM wortmannin for 10 min and stimulated with 100 nM SDF-1 for 10 min. (B) The cells were stimulated with 100 nM SDF-1 for 1, 5, 10, and 15 min. Where indicated, 100 nM wortmannin was added 5 min before termination of the stimulation. Activated (pSer473PKB) and total protein kinase B (PKB) were determined as in the legend to Fig. 1 A. (C) Protein kinase B activity in immunoprecipitates. The cells were either treated with medium (Cont), 100 nM SDF-1 (SDF-1), or 100 nM wortmannin (wort) for 10 min, or were treated with 100 nM wortmannin for 10 min before stimulation with 100 nM SDF-1 for 10 min (wort > SDF-1), or were stimulated with 100 nM SDF-1 and 5 min later treated with 100 nM wortmannin for an additional 5 min (SDF-1 > wort). Results are expressed as in Fig. 1 C. D. PIP3 formation in [32P]orthophosphate-loaded T cells. The cells were either treated with medium (Cont), 100 nM SDF-1 (SDF-1 10′), 100 nM wortmannin (wort), 10 µM SDF-1P2G (P2G) for 10 min or with 100 nM SDF-1 for 1 min (SDF-1 1′), or were stimulated with 100 nM SDF-1 and 5 min later treated with 100 nM wortmannin (SDF-1 > wort) or 10 μM SDF-1P2G (SDF-1 > P2G) for an additional 5 min. Lipids were extracted, deacylated, and glycerophosphoinositides were separated by HPLC (duplicate determinations). Results are representative for at least three independent experiments.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
9.
Figure 6

Figure 6. From: Signal Transduction by Cxc Chemokine Receptor 4.

Restimulation of CXCR4 with SDF-1. (A) Protein kinase B activation. T cells were stimulated with 100 nM SDF-1 for the indicated times (points connected by solid line). In parallel samples, a second addition of 100 nM SDF-1 was made at 5, 20, or 60 min of stimulation (arrowheads), and the incubation continued for 1 min (points connected by thin line). The cells were lysed, and protein kinase B activity was measured in immunoprecipitates. Maximum protein kinase B activity obtained at 1 min of stimulation was set as 100%. (B) Changes in intracellular free calcium. Fluorescence of Fura-2–loaded T cells was recorded for 6 min in parallel samples. At time 0 the cells were stimulated with 80 nM SDF-1. Where indicated, 1 μM SDF-1P2G (P2G) was added 90 s later to one sample (bottom trace), and at ∼280 s a second addition of 1 μM SDF-1 (SDF-1) was made to both samples. (Inset) 1 μM SDF-1P2G (P2G) does not desensitize the response to 1 μM SDF-1 (SDF-1). Results are representative for at least three independent experiments performed in duplicate with cells from different donors.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
10.
Figure 1

Figure 1. From: Signal Transduction by Cxc Chemokine Receptor 4.

Chemokine-stimulated activation of protein kinase B and ERK-2 in T cells. (A) Activation of protein kinase B determined by Western blot analysis. T cells were stimulated with 200 nM MCP-1, MIP-1β, LARC, ELC, IP10, or SDF-1 for the indicated times. Whole cell lysates were separated on SDS-PAGE and activated protein kinase B was detected with an antibody reacting with phosphorylated Ser473 (pS47 3PKB, left). To confirm equal loading of protein kinase B, blots reprobed with an antibody reacting with both activated and nonactivated protein kinase B (PKB, right). (B) Time course of protein kinase B activation. Enzyme activity was determined in immunoprecipitates from lysates of cells stimulated with 100 nM SDF-1. Kinase assays were performed in duplicate, and the results are presented as fold activation (mean values). (C) Comparison of protein kinase B activity in immunoprecipitates from cells stimulated for 1 or 5 min with 100 nM SDF-1, ELC, MCP-1, or MIP-1β. (D) Western blot analysis of ERK-2 activation. Cells were stimulated with 200 nM MCP-1, MIP-1β, LARC, ELC, IP10, or SDF-1 for the indicated times and whole cells lysates were separated by SDS-PAGE. Activated ERK-2 (pERK-2) migrates with a slower electrophoretic mobility than nonactivated ERK-2 (ERK-2). Representative results obtained with cells from single donors are shown. Independent experiments with at least four more batches of cells from different donors gave similar results.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
11.
Figure 1

Figure 1. From: Signal Transduction by Cxc Chemokine Receptor 4.

Chemokine-stimulated activation of protein kinase B and ERK-2 in T cells. (A) Activation of protein kinase B determined by Western blot analysis. T cells were stimulated with 200 nM MCP-1, MIP-1β, LARC, ELC, IP10, or SDF-1 for the indicated times. Whole cell lysates were separated on SDS-PAGE and activated protein kinase B was detected with an antibody reacting with phosphorylated Ser473 (pS47 3PKB, left). To confirm equal loading of protein kinase B, blots reprobed with an antibody reacting with both activated and nonactivated protein kinase B (PKB, right). (B) Time course of protein kinase B activation. Enzyme activity was determined in immunoprecipitates from lysates of cells stimulated with 100 nM SDF-1. Kinase assays were performed in duplicate, and the results are presented as fold activation (mean values). (C) Comparison of protein kinase B activity in immunoprecipitates from cells stimulated for 1 or 5 min with 100 nM SDF-1, ELC, MCP-1, or MIP-1β. (D) Western blot analysis of ERK-2 activation. Cells were stimulated with 200 nM MCP-1, MIP-1β, LARC, ELC, IP10, or SDF-1 for the indicated times and whole cells lysates were separated by SDS-PAGE. Activated ERK-2 (pERK-2) migrates with a slower electrophoretic mobility than nonactivated ERK-2 (ERK-2). Representative results obtained with cells from single donors are shown. Independent experiments with at least four more batches of cells from different donors gave similar results.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.
12.
Figure 1

Figure 1. From: Signal Transduction by Cxc Chemokine Receptor 4.

Chemokine-stimulated activation of protein kinase B and ERK-2 in T cells. (A) Activation of protein kinase B determined by Western blot analysis. T cells were stimulated with 200 nM MCP-1, MIP-1β, LARC, ELC, IP10, or SDF-1 for the indicated times. Whole cell lysates were separated on SDS-PAGE and activated protein kinase B was detected with an antibody reacting with phosphorylated Ser473 (pS47 3PKB, left). To confirm equal loading of protein kinase B, blots reprobed with an antibody reacting with both activated and nonactivated protein kinase B (PKB, right). (B) Time course of protein kinase B activation. Enzyme activity was determined in immunoprecipitates from lysates of cells stimulated with 100 nM SDF-1. Kinase assays were performed in duplicate, and the results are presented as fold activation (mean values). (C) Comparison of protein kinase B activity in immunoprecipitates from cells stimulated for 1 or 5 min with 100 nM SDF-1, ELC, MCP-1, or MIP-1β. (D) Western blot analysis of ERK-2 activation. Cells were stimulated with 200 nM MCP-1, MIP-1β, LARC, ELC, IP10, or SDF-1 for the indicated times and whole cells lysates were separated by SDS-PAGE. Activated ERK-2 (pERK-2) migrates with a slower electrophoretic mobility than nonactivated ERK-2 (ERK-2). Representative results obtained with cells from single donors are shown. Independent experiments with at least four more batches of cells from different donors gave similar results.

Bettina Tilton, et al. J Exp Med. 2000 August 7;192(3):313-324.

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