American Society of Hematology

Figure 3.

$Figure 3. CD19 tyrosine phosphorylation induced by LPS stimulation and anti-RP105 ligation. (A-B) Wild-type A20 cells were incubated with (A) LPS or (B) anti-RP105 Abs for the indicated times or anti-IgG Abs for 3 minutes. In panel A, cells pretreated with anti–MD-1 Abs were also assessed. Proteins immunoprecipitated by anti-CD19 were fractionated by SDS-PAGE and transferred onto nitrocellulose for subsequent antiphosphotyrosine (pTyr), anti-Lyn, anti-Vav, anti–PI 3-kinase, and anti-CD19 immunoblotting. (C) CD19 phosphorylation levels were quantified with background and maximum phosphorylation levels defined as 0% and 100%, respectively (mean % ± SEM). (D) Splenic B cells from a C57BL/6 mouse were incubated with LPS, anti-RP105, or anti-IgM Abs for indicated times. Cell lysates were subjected to SDS-PAGE and transferred onto a membrane for anti-phosphoCD19 immunoblotting. The membrane was reprobed with anti-CD19 Abs. (E) Splenic B cells from C3H/HeN and C3H/HeJ mice were incubated with LPS and processed as in panel D. (F) Lipid rafts were isolated by lysis of cells in 0.05% Triton X-100 at 4°C and flotation on a discontinuous sucrose gradient. Fractions were collected and subjected to CD19 or RP105 immunoprecipitation and SDS-PAGE. Fractions 4 to 6 and 10 to 12 represent the raft containing fractions and the soluble protein fractions. To detect CD19 or RP105, cell surface was biotinylated prior to anti-RP105 stimulation, and the membrane was blotted using HRP-conjugated neutroavidin. Lysates were also subjected to SDS-PAGE and immunoblotting with anti-Lyn and anti-CD45R to confirm the proper preparation of lipid rafts as well as the equivalent amounts between the samples. These results are representative of those obtained in 3 independent experiments.$

CD19 tyrosine phosphorylation induced by LPS stimulation and anti-RP105 ligation. (A-B) Wild-type A20 cells were incubated with (A) LPS or (B) anti-RP105 Abs for the indicated times or anti-IgG Abs for 3 minutes. In panel A, cells pretreated with anti–MD-1 Abs were also assessed. Proteins immunoprecipitated by anti-CD19 were fractionated by SDS-PAGE and transferred onto nitrocellulose for subsequent antiphosphotyrosine (pTyr), anti-Lyn, anti-Vav, anti–PI 3-kinase, and anti-CD19 immunoblotting. (C) CD19 phosphorylation levels were quantified with background and maximum phosphorylation levels defined as 0% and 100%, respectively (mean % ± SEM). (D) Splenic B cells from a C57BL/6 mouse were incubated with LPS, anti-RP105, or anti-IgM Abs for indicated times. Cell lysates were subjected to SDS-PAGE and transferred onto a membrane for anti-phosphoCD19 immunoblotting. The membrane was reprobed with anti-CD19 Abs. (E) Splenic B cells from C3H/HeN and C3H/HeJ mice were incubated with LPS and processed as in panel D. (F) Lipid rafts were isolated by lysis of cells in 0.05% Triton X-100 at 4°C and flotation on a discontinuous sucrose gradient. Fractions were collected and subjected to CD19 or RP105 immunoprecipitation and SDS-PAGE. Fractions 4 to 6 and 10 to 12 represent the raft containing fractions and the soluble protein fractions. To detect CD19 or RP105, cell surface was biotinylated prior to anti-RP105 stimulation, and the membrane was blotted using HRP-conjugated neutroavidin. Lysates were also subjected to SDS-PAGE and immunoblotting with anti-Lyn and anti-CD45R to confirm the proper preparation of lipid rafts as well as the equivalent amounts between the samples. These results are representative of those obtained in 3 independent experiments.

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