Figure 6. Panel A shows flow cytometry... | Rockefeller University Press

Figure 6.

Panel A shows flow cytometry histograms comparing pAKT-T308 levels in TACI KO CD45.2 positive, WT CD45.2 positive, and WT CD45.1 positive resting MZ B cells at 37 degrees Celsius from WT:WT and KO:WT chimeras. Red represents TACI KO CD45.2 positive MZ, black represents WT CD45.2 positive MZ, grey represents WT CD45.1 positive. Panel B is a dot plot showing pAKT-T308 mean fluorescence intensity (MFI) measured by flow cytometry in CD45.2 positive and CD45.1 positive resting MZ B cells from WT:WT and KO:WT chimeras. WT:WT n equals 4 and KO:WT n equals 5. Lines connect cells from the same sample. Black represents WT CD45.2 positive, red represents TACI KO CD45.2 positive, and grey represents WT CD45.1 positive. Panel C shows flow cytometry histograms comparing pAKT-S473 levels in TACI KO CD45.2 positive, WT CD45.2 positive, and WT CD45.1 positive resting MZ B cells at 37 degrees Celsius from WT:WT and KO:WT chimeras. Red represents TACI KO CD45.2 positive MZ, black represents WT CD45.2 positive MZ, grey represents WT CD45.1 positive, and dashed lines represent cells treated with the PI3K inhibitor idelalisib. Panel D is a dot plot showing pAKT-S473 MFI measured by flow cytometry in CD45.2 positive and CD45.1 positive resting MZ B cells from WT:WT and KO:WT chimeras; n equals 4. Lines connect cells from the same sample. Black represents WT CD45.2 positive, red represents TACI KO CD45.2 positive, and grey represents WT CD45.1 positive. Panel E shows flow cytometry plots and histograms comparing pS6-S235 slash 236 levels in WT and TACI KO CD45.2 positive resting MZ B cells at 37 degrees Celsius from WT:WT and KO:WT chimeras. Black represents WT CD45.2 positive, red represents TACI KO CD45.2 positive, and grey represents WT cells stained with isotype control antibody. Panel F shows dot plots showing the percentage of pS6 positive cells and pS6 MFI within pS6 positive cells measured by flow cytometry in CD45.2 positive and CD45.1 positive resting MZ B cells from WT:WT and KO:WT chimeras; n equals 5. Lines connect cells from the same sample. Black represents WT CD45.2 positive, red represents TACI KO CD45.2 positive, and grey represents WT CD45.1 positive. Panel G shows an immunoblot of anti-TACI immunoprecipitation from LPS (lipopolysaccharide)-activated B cells, alongside a control immunoprecipitation (IP) using isotype-control antibody and the input cell lysates. Cells were stimulated with APRIL (A Proliferation-Inducing Ligand), BAFF (B cell Activating Factor), or media-only control for 15 minutes at 37 degrees Celsius. The immunoblot was probed with anti-TACI, anti-p110 (phosphoinositide 3-kinase catalytic subunit), anti-p85 (phosphoinositide 3-kinase regulatory subunit), anti-mTOR (mechanistic Target Of Rapamycin), and anti-MyD88 (Myeloid Differentiation Primary Response 88). The same membrane sections are shown at two different image intensities for p110, p85, and mTOR due to higher protein levels in the input lysates.

AKT-mTORC1 signaling is impaired in TACI KO MZ B cells. (A) Flow cytometry histograms comparing pAKT-T308 levels in TACI KO CD45.2⁺, WT CD45.2⁺, and WT CD45.1⁺ resting MZ B cells at 37°C from WT:WT and KO:WT chimeras. Red: TACI KO CD45.2⁺ MZ; black: WT CD45.2⁺ MZ; gray: WT CD45.1⁺; dashed lines: cells treated with the PI3Kδ inhibitor idelalisib (PI3Ki). (B) pAKT-T308 MFI, measured by flow cytometry, in CD45.2⁺ and CD45.1⁺ resting MZ B cells from WT:WT and KO:WT chimeras. WT:WT, n = 4; and KO:WT, n = 5. Lines connect cells from the same sample. Black: WT CD45.2⁺; red: TACI KO CD45.2⁺; gray: WT CD45.1⁺. (C) Flow cytometry histograms comparing pAKT-S473 levels in TACI KO CD45.2⁺, WT CD45.2⁺, and WT CD45.1⁺ resting MZ B cells at 37°C from WT:WT and KO:WT chimeras. Red: TACI KO CD45.2⁺ MZ; black: WT CD45.2⁺ MZ; gray: WT CD45.1⁺; dashed lines: cells treated with the PI3Kδ inhibitor idelalisib (PI3Ki). (D) pAKT-S473 MFI, measured by flow cytometry, in CD45.2⁺ and CD45.1⁺ resting MZ B cells from WT:WT and KO:WT chimeras; n = 4. Lines connect cells from the same sample. Black: WT CD45.2⁺; red: TACI KO CD45.2⁺; gray: WT CD45.1⁺. (E) Flow cytometry plots and histograms comparing pS6-S235/236 levels in WT and TACI KO CD45.2⁺ resting MZ B cells at 37°C from WT:WT and KO:WT chimeras. Black: WT CD45.2⁺; red: TACI KO CD45.2⁺; gray: WT cells stained with isotype control antibody (Isotype). (F) Percentage of pS6⁺ cells (left) and pS6 MFI within pS6⁺ cells (right), measured by flow cytometry, in CD45.2⁺ and CD45.1⁺ resting MZ B cells from WT:WT and KO:WT chimeras; n = 5. Lines connect cells from the same sample. Black: WT CD45.2⁺; red: TACI KO CD45.2⁺; gray: WT CD45.1⁺. (G) Immunoblot anti-TACI immunoprecipitation (IP) from LPS-activated B cells, alongside a control IP using isotype control antibody (IC) and the input cell lysates. Cells were stimulated with APRIL, BAFF, or media-only control for 15 min at 37°C. The immunoblot was probed with anti-TACI, anti-p110δ, anti-p85α, anti-mTOR, and anti-MyD88. The same membrane sections are displayed at two different image intensities for p110δ, p85α, and mTOR due to higher protein levels in the input lysates. Statistical test: repeated-measures two-way ANOVA with Fisher’s LSD test (B, D, and F). Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. Data are from one of four (B and F) or two (D and G) independent experiments. Source data are available for this figure: SourceData F6.