Figure 6
Figure 6. DM-induced phosphorylation of Lck is CD45-dependent event. (A) CD45 specific phosphatase activity was assayed from the control, CXCL12-treated, and DM-treated Jurkat cells using a kit manufactured by BIOMOL International. Jurkat cells were treated with CXCL12 (100 ng/mL) and DM (1 μM) alone or in combination for 5 minutes and after which the cells were lysed with a phosphate-free lysis buffer supplied by the manufacturer. CD45 was immunoprecipitated using monoclonal antibody after which the phosphatase activity was assayed. A phosphoprotein was used as a substrate with total reaction volume 100 μL over a 30-minute incubation period in which 50 μg of experimental sample was added. The activity was calculated from an inorganic phosphate standard curve. Finally, the specific phosphatase activity was expressed in nmol phosphate/minutes per mg of protein. In addition, the presence of CD45 in the lysate was confirmed by immunoblot and is shown as an insert in panel A. (B) Wild-type Jurkat cells and the CD45-deficient Jurkat cell line (J45.01) were treated with CXCL12 (100 ng/mL) and/or DM (1 μM) for 5 minutes or after pretreatment with CD45 blocker, RK-682 (25 μM) for 30 minutes followed by treatment with CXCL12 and/or DM for 5 minutes. CD45-deficient cell line was used as negative control for the experiment. Immunoprecipitation and immunoblotting of phospho-Lck and total Lck were performed, and the data are presented as described in Figure 1A. It should be noted that the ability of DM to inhibit the serine phosphorylation of Lck (shown in Figure S1B) suggests that the lower band noted as IgG in these gels may also be a different gel migrating form of phosphorylated Lck. (C) Primary human T cells, wild-type Jurkat T cells (JE6.1), and Jurkat cells deficient for Lck (JCaM1.6) and CD45 (J45.01) cells were labeled with calcein and treated with 1 μM DM or vehicle for 2 hours, after which CXCL12 migration assays. Data represented the average value of 3 individual experiments plus or minus SE. PMI indicates the percent migration index.

DM-induced phosphorylation of Lck is CD45-dependent event. (A) CD45 specific phosphatase activity was assayed from the control, CXCL12-treated, and DM-treated Jurkat cells using a kit manufactured by BIOMOL International. Jurkat cells were treated with CXCL12 (100 ng/mL) and DM (1 μM) alone or in combination for 5 minutes and after which the cells were lysed with a phosphate-free lysis buffer supplied by the manufacturer. CD45 was immunoprecipitated using monoclonal antibody after which the phosphatase activity was assayed. A phosphoprotein was used as a substrate with total reaction volume 100 μL over a 30-minute incubation period in which 50 μg of experimental sample was added. The activity was calculated from an inorganic phosphate standard curve. Finally, the specific phosphatase activity was expressed in nmol phosphate/minutes per mg of protein. In addition, the presence of CD45 in the lysate was confirmed by immunoblot and is shown as an insert in panel A. (B) Wild-type Jurkat cells and the CD45-deficient Jurkat cell line (J45.01) were treated with CXCL12 (100 ng/mL) and/or DM (1 μM) for 5 minutes or after pretreatment with CD45 blocker, RK-682 (25 μM) for 30 minutes followed by treatment with CXCL12 and/or DM for 5 minutes. CD45-deficient cell line was used as negative control for the experiment. Immunoprecipitation and immunoblotting of phospho-Lck and total Lck were performed, and the data are presented as described in Figure 1A. It should be noted that the ability of DM to inhibit the serine phosphorylation of Lck (shown in Figure S1B) suggests that the lower band noted as IgG in these gels may also be a different gel migrating form of phosphorylated Lck. (C) Primary human T cells, wild-type Jurkat T cells (JE6.1), and Jurkat cells deficient for Lck (JCaM1.6) and CD45 (J45.01) cells were labeled with calcein and treated with 1 μM DM or vehicle for 2 hours, after which CXCL12 migration assays. Data represented the average value of 3 individual experiments plus or minus SE. PMI indicates the percent migration index.

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