American Society of Hematology

Figure 1

$Figure 1. In vitro phosphatase assay showing increased CD45RB phosphatase activity and translocation of CD45RB to lipid rafts. (A) CD45RB immunoprecipitated from PBMC lysates treated with anti-CD45RB mAb (6G3) treated at various time points. Cells were lysed in lysis buffer without any phosphatase inhibitors. Lck, immunoprecipitated from untreated cells lysed in buffer containing phosphatase inhibitors. The last lane of pTyr blot shows phosphorylated Lck in resting cells. Immunoprecipitated CD45RB was then incubated with immunprecipitated Lck in a phosphatase reaction buffer for 20 minutes at 30°C. SDS sample buffer was added, and the samples were boiled and run on an 8% SDS-PAGE gel, transferred, and probed with a phospho-tyrosine Ab. Reduced tyrosine phosphorylation of Lck observed over time compared with phosphorylated Lck in resting cells. (B) Tyrosine 505 on Lck shows a decrease in phosphorylation over a period of 20 minutes after treatment of whole-cell lysates of mononuclear cells with anti-CD45RB mAb (6G3). (C) Pooled raft (R) and nonraft (NR) fractions of control and anti-CD45RB (6G3) mAb-treated cells as obtained by sucrose density gradient show translocation of CD45RB to the raft fraction of treated cells. We also stimulated using antimouse IgG1, which was used as an isotypic control where CD45RB is found only in the nonraft fractions.$

In vitro phosphatase assay showing increased CD45RB phosphatase activity and translocation of CD45RB to lipid rafts. (A) CD45RB immunoprecipitated from PBMC lysates treated with anti-CD45RB mAb (6G3) treated at various time points. Cells were lysed in lysis buffer without any phosphatase inhibitors. Lck, immunoprecipitated from untreated cells lysed in buffer containing phosphatase inhibitors. The last lane of pTyr blot shows phosphorylated Lck in resting cells. Immunoprecipitated CD45RB was then incubated with immunprecipitated Lck in a phosphatase reaction buffer for 20 minutes at 30°C. SDS sample buffer was added, and the samples were boiled and run on an 8% SDS-PAGE gel, transferred, and probed with a phospho-tyrosine Ab. Reduced tyrosine phosphorylation of Lck observed over time compared with phosphorylated Lck in resting cells. (B) Tyrosine 505 on Lck shows a decrease in phosphorylation over a period of 20 minutes after treatment of whole-cell lysates of mononuclear cells with anti-CD45RB mAb (6G3). (C) Pooled raft (R) and nonraft (NR) fractions of control and anti-CD45RB (6G3) mAb-treated cells as obtained by sucrose density gradient show translocation of CD45RB to the raft fraction of treated cells. We also stimulated using antimouse IgG₁, which was used as an isotypic control where CD45RB is found only in the nonraft fractions.

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