Figure 4. B cell–intrinsic ITK-SYK expression does not affect B cell development or activation. (A) Single cell suspensions from the spleens or peritoneal cavities of ITK-SYKCD19-Cre or control (CD19-Cre) mice were stained against B220, CD21, CD23, and CD5. eGFP fluorescence indicative of ITK-SYK expression was determined in the splenic follicular (FO), splenic marginal zone (MZ), or peritoneal B1 B cell compartments using FACS. (B) Cytoplasmic extracts of purified mature B cells from 5-wk-old control or ITK-SYKCD19-Cre mice were subjected to Western blot analysis with an antibody against phospho–tyrosine (p-Tyr). Western blotting for β-actin demonstrated equal protein loading. Data shown are representative of three independent experiments. (C) Total splenic B or T cell numbers from ITK-SYKCD19-Cre (n = 12) or control (n = 6) mice are shown. Each symbol represents an individual mouse. Statistical significance was analyzed using the unpaired two-tailed Student’s t test. ns, non–statistically significant differences (P ≥ 0.05). Horizontal bars indicate the means. (D) Lymphocyte preparations from bone marrow (BM) or spleen (SPL) of ITK-SYKCD19-Cre or control mice were stained against B220, IgM, CD21, or CD23 and analyzed by FACS. The percentages of individual B cell populations are indicated. FO, follicular B cells; MZ, marginal zone B cells. (E) Forward scattering (FCS) and expression of CD86 was analyzed on B220+ mature B cells from ITK-SYKCD19-Cre or control mice using FACS. Data shown in A and C–E are representative of four independent experiments with total numbers of at least six 5–7-wk-old mice per genotype analyzed. (F) ITK-SYK is constitutively associated with T but not with B cell lipid rafts. Lipid raft and nonraft fractions were prepared from purified splenic T cells of control or ITK-SYKCD4-Cre mice or from mature B cells of ITK-SYKCD19-Cre mice that were >12 wk old. The fractions were subsequently analyzed for the presence of ITK or ITK-SYK by Western blot analysis using an antibody against ITK. Dot blots for GM1 show successful raft preparation and equal loading. Data shown are representative of three independent experiments.
Figure 4.

B cell–intrinsic ITK-SYK expression does not affect B cell development or activation. (A) Single cell suspensions from the spleens or peritoneal cavities of ITK-SYKCD19-Cre or control (CD19-Cre) mice were stained against B220, CD21, CD23, and CD5. eGFP fluorescence indicative of ITK-SYK expression was determined in the splenic follicular (FO), splenic marginal zone (MZ), or peritoneal B1 B cell compartments using FACS. (B) Cytoplasmic extracts of purified mature B cells from 5-wk-old control or ITK-SYKCD19-Cre mice were subjected to Western blot analysis with an antibody against phospho–tyrosine (p-Tyr). Western blotting for β-actin demonstrated equal protein loading. Data shown are representative of three independent experiments. (C) Total splenic B or T cell numbers from ITK-SYKCD19-Cre (n = 12) or control (n = 6) mice are shown. Each symbol represents an individual mouse. Statistical significance was analyzed using the unpaired two-tailed Student’s t test. ns, non–statistically significant differences (P ≥ 0.05). Horizontal bars indicate the means. (D) Lymphocyte preparations from bone marrow (BM) or spleen (SPL) of ITK-SYKCD19-Cre or control mice were stained against B220, IgM, CD21, or CD23 and analyzed by FACS. The percentages of individual B cell populations are indicated. FO, follicular B cells; MZ, marginal zone B cells. (E) Forward scattering (FCS) and expression of CD86 was analyzed on B220+ mature B cells from ITK-SYKCD19-Cre or control mice using FACS. Data shown in A and C–E are representative of four independent experiments with total numbers of at least six 5–7-wk-old mice per genotype analyzed. (F) ITK-SYK is constitutively associated with T but not with B cell lipid rafts. Lipid raft and nonraft fractions were prepared from purified splenic T cells of control or ITK-SYKCD4-Cre mice or from mature B cells of ITK-SYKCD19-Cre mice that were >12 wk old. The fractions were subsequently analyzed for the presence of ITK or ITK-SYK by Western blot analysis using an antibody against ITK. Dot blots for GM1 show successful raft preparation and equal loading. Data shown are representative of three independent experiments.

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