Figure 3.
Figure 3. SHIP1 inhibits LPS-induced activation of MAPKs primarily via a phosphatase activity- and PI-3K-independent mechanism in macrophages. (A) LY294002 affects LPS-induced activation of MAPKs in macrophages. RAW264.7 macrophages were pretreated with 30 μM LY303511 or 30 μM LY294002 for 40 minutes and then stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK were detected by Western blot. Phosphorylation levels of the proteins at the time point of 20 minutes after LPS stimulation were quantitated by measuring band intensities. The values were normalized to the total ERK, p38, or JNK signal, respectively. Relative phosphorylation levels of the proteins in the control cells were expressed as 1. (B) Overexpression of wild-type SHIP1 or phosphatase activity-disrupted mutant SHIP1 inhibits LPS-induced activation of MAPKs in macrophages. RAW264.7 macrophages were transiently transfected with control plasmid, wild-type SHIP1, or mutant SHIP1 and cultured for 24 hours. The cells were stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK were detected and shown as described in panel A. (C-D) Overexpression of wild-type SHIP1 or phosphatase activity-disrupted mutant SHIP1 inhibits LPS-induced activation of MAPKs in the presence of PI-3K inhibitor in macrophages. Control plasmid, wild-type SHIP1, or phosphatase-inactive mutant SHIP1-transfected RAW264.7 macrophages were pretreated with (C) 30 μM LY294002 (LY) or (D) 100 nM wortmannin (W) for 40 minutes and then stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK was detected and shown as described in panel A. (E) SHIP1 RNA interfering enhances LPS-induced activation of MAPKs in macrophages. RAW264.7 macrophages were transfected with pU6-SHIP1 or control plasmid and cultured for 48 hours. The cells were stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK were detected and shown as described in panel A. (F) SHIP1 RNA interfering enhances LPS-induced activations of MAPKs in the presence of LY294002 in macrophages. RAW264.7 macrophages were transfected and cultured as described in panel E. All of the cells were pretreated with 30 μM LY294002 for 40 minutes and then stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK were detected by Western blotting, densitometrically analyzed, and presented as described in panel A. Similar results were observed in 3 separate experiments.

SHIP1 inhibits LPS-induced activation of MAPKs primarily via a phosphatase activity- and PI-3K-independent mechanism in macrophages. (A) LY294002 affects LPS-induced activation of MAPKs in macrophages. RAW264.7 macrophages were pretreated with 30 μM LY303511 or 30 μM LY294002 for 40 minutes and then stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK were detected by Western blot. Phosphorylation levels of the proteins at the time point of 20 minutes after LPS stimulation were quantitated by measuring band intensities. The values were normalized to the total ERK, p38, or JNK signal, respectively. Relative phosphorylation levels of the proteins in the control cells were expressed as 1. (B) Overexpression of wild-type SHIP1 or phosphatase activity-disrupted mutant SHIP1 inhibits LPS-induced activation of MAPKs in macrophages. RAW264.7 macrophages were transiently transfected with control plasmid, wild-type SHIP1, or mutant SHIP1 and cultured for 24 hours. The cells were stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK were detected and shown as described in panel A. (C-D) Overexpression of wild-type SHIP1 or phosphatase activity-disrupted mutant SHIP1 inhibits LPS-induced activation of MAPKs in the presence of PI-3K inhibitor in macrophages. Control plasmid, wild-type SHIP1, or phosphatase-inactive mutant SHIP1-transfected RAW264.7 macrophages were pretreated with (C) 30 μM LY294002 (LY) or (D) 100 nM wortmannin (W) for 40 minutes and then stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK was detected and shown as described in panel A. (E) SHIP1 RNA interfering enhances LPS-induced activation of MAPKs in macrophages. RAW264.7 macrophages were transfected with pU6-SHIP1 or control plasmid and cultured for 48 hours. The cells were stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK were detected and shown as described in panel A. (F) SHIP1 RNA interfering enhances LPS-induced activations of MAPKs in the presence of LY294002 in macrophages. RAW264.7 macrophages were transfected and cultured as described in panel E. All of the cells were pretreated with 30 μM LY294002 for 40 minutes and then stimulated with 100 ng/mL LPS for the indicated time periods. The phosphorylation of ERK1/2, p38, and JNK were detected by Western blotting, densitometrically analyzed, and presented as described in panel A. Similar results were observed in 3 separate experiments.

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