Figure 1.
Constitutively active B-cell–specific Lyn enhances signaling upon BCR ligation in normal B cells. (A) Schematic overview of the targeting strategy to establish the LynY508F-IRES-GFP conditional transgene mice using recombinase-mediated cassette exchange (RMCE). (B) Flow cytometric analysis of GFP expression of CD19+ B cells in the spleen of WT and Lynup-B mice. (C) Western blot analysis of purified B cells isolated from spleens of WT and Lynup-B mice. Cells were kept untreated or stimulated with 20 mg/mL IgM for 10 minutes before lysis. The densitometry of the phospho-Syk and phospho-Ship1 bands was normalized to the densitometry of the corresponding β-actin bands. The graph shows the fold changes in densitometry of unstimulated to IgM-stimulated B cells from the same mouse (including samples shown in supplemental Figure 1D). (D) Tyrosine phosphorylation profile of purified B cells from WT (n = 3) and Lynup-B (n = 3) mice upon IgM stimulation. Kinases in B-cell lysates actively phosphorylated substrates on the PamChip. Tyrosine phosphorylation was detected by a FITC-conjugated PY20 antibody to quantify the phosphorylation signal. LFC values between untreated and IgM-stimulated samples were calculated. Each column of the heatmap represents the mean LFC of 3 mice per genotype. A red row indicates higher phosphorylation of the peptides upon IgM treatment, and a blue row implies a lower phosphorylation after IgM treatment. Supplemental Table 4 provides the LFC values for each phosphorylated peptide on the PamChip. (E) Dephosphorylation profile of purified B cells upon IgM stimulation in WT (n = 2) and Lynup-B (n = 2) mice. Phosphatases in the B-cell lysates dephosphorylated proprietary nitrophosphotyrosine residues on a chip. Unphosphorylated nitrotyrosine residues were detected by an anti-nitrotyrosine antibody. LFC values between untreated and IgM stimulated samples were calculated. Each column of the heatmap represents the mean LFC of 2 mice per genotype. A red row indicates higher dephosphorylation of the peptides after IgM treatment, and a blue row implies a lower dephosphorylation after IgM treatment. Supplemental Table 5 provides the LFC values for each dephosphorylated peptide on the phosphatase chip. FSC-A, forward scatter-area; FSC-H, forward scatter-height; SA, splice acceptor sequence.

Constitutively active B-cell–specific Lyn enhances signaling upon BCR ligation in normal B cells. (A) Schematic overview of the targeting strategy to establish the LynY508F-IRES-GFP conditional transgene mice using recombinase-mediated cassette exchange (RMCE). (B) Flow cytometric analysis of GFP expression of CD19+ B cells in the spleen of WT and Lynup-B mice. (C) Western blot analysis of purified B cells isolated from spleens of WT and Lynup-B mice. Cells were kept untreated or stimulated with 20 mg/mL IgM for 10 minutes before lysis. The densitometry of the phospho-Syk and phospho-Ship1 bands was normalized to the densitometry of the corresponding β-actin bands. The graph shows the fold changes in densitometry of unstimulated to IgM-stimulated B cells from the same mouse (including samples shown in supplemental Figure 1D). (D) Tyrosine phosphorylation profile of purified B cells from WT (n = 3) and Lynup-B (n = 3) mice upon IgM stimulation. Kinases in B-cell lysates actively phosphorylated substrates on the PamChip. Tyrosine phosphorylation was detected by a FITC-conjugated PY20 antibody to quantify the phosphorylation signal. LFC values between untreated and IgM-stimulated samples were calculated. Each column of the heatmap represents the mean LFC of 3 mice per genotype. A red row indicates higher phosphorylation of the peptides upon IgM treatment, and a blue row implies a lower phosphorylation after IgM treatment. Supplemental Table 4 provides the LFC values for each phosphorylated peptide on the PamChip. (E) Dephosphorylation profile of purified B cells upon IgM stimulation in WT (n = 2) and Lynup-B (n = 2) mice. Phosphatases in the B-cell lysates dephosphorylated proprietary nitrophosphotyrosine residues on a chip. Unphosphorylated nitrotyrosine residues were detected by an anti-nitrotyrosine antibody. LFC values between untreated and IgM stimulated samples were calculated. Each column of the heatmap represents the mean LFC of 2 mice per genotype. A red row indicates higher dephosphorylation of the peptides after IgM treatment, and a blue row implies a lower dephosphorylation after IgM treatment. Supplemental Table 5 provides the LFC values for each dephosphorylated peptide on the phosphatase chip. FSC-A, forward scatter-area; FSC-H, forward scatter-height; SA, splice acceptor sequence.

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