American Society of Hematology

Figure 1.

$Inpp5k inactivation results in altered IL-7R signaling and Pax5 expression in fractions A and B of the bone marrow. (A) PAX5 protein expression was analyzed using flow cytometry after plasma and nuclear membrane permeabilization of control (WT, red areas and columns) and VAV-CRE (blue areas and columns) cells from fractions A and B. Representative MFI histograms of PAX5 protein expression (left). Quantified expression of PAX5 protein after MFI normalization to WT with mean MFI (right). Results represent individual mice (n = 8-9 mice per group) and mean ± SEM. (B) Flow cytometry analysis of p-AKTS473 in plasma membrane–permeabilized cells from fractions A and B isolated from control (WT, red lines) and VAV-CRE (blue lines) mice, before (0) and 2 to 10 minutes after ex vivo addition of IL-7 (2 ng/mL) at 37°C. Quantitative expression of p-AKTS473 normalized to WT with mean MFI at t = 0 (before IL-7 addition). Results are representative of 2 independent experiments for a total of 6 mice. (C) EBF1 protein expression was analyzed using flow cytometry after plasma and nuclear membrane permeabilization of control (WT, red areas and columns) and VAV-CRE (blue areas and columns) cells from fractions A and B. Representative MFI histograms of EBF1 protein expression (top). The quantitative expression of EBF1 protein after MFI normalization to WT with mean MFI (bottom). Results represent individual mice (n = 5-9 mice per group) and mean ± SEM. (D) Flow cytometry analysis of p-STAT5Y694 in plasma membrane–permeabilized cells from fractions A and B isolated from control (WT, red lines) and VAV-CRE (blue lines) mice, before (0) and 2 to 10 minutes after the ex vivo addition of IL-7 (2 ng/mL) at 37°C. Quantitative expression of p-STAT5Y694 normalized to WT with mean MFI at t = 0 (before IL-7 addition). Results are representative of a total of 6 mice. (E-F) Flow cytometry analysis of p-JAK1Y1022 (E) and p-JAK3Y980/981 (F) in plasma membrane–permeabilized cells from factions A and B isolated from control (WT, red lines) and VAV-CRE (blue lines) mice, before (0), 0.5, 1, 2, and 10 minutes after the ex vivo addition of IL-7 (2 ng/mL) at 37°C. Quantified expression of p-JAK1Y1022 (E) and p-JAK3Y980/981 (F) normalized to WT MFI at t = 0 (before IL-7 addition). Results are representative of 4 independent experiments for a total of 8 to 12 mice. P values were calculated using unpaired nonparametric t test. NS: P > .05; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. NS, nonsignificant; MFI, mean fluorescence intensity; SEM, standard error of the mean; WT, wild type.$

Inpp5k inactivation results in altered IL-7R signaling and Pax5 expression in fractions A and B of the bone marrow. (A) PAX5 protein expression was analyzed using flow cytometry after plasma and nuclear membrane permeabilization of control (WT, red areas and columns) and VAV-CRE (blue areas and columns) cells from fractions A and B. Representative MFI histograms of PAX5 protein expression (left). Quantified expression of PAX5 protein after MFI normalization to WT with mean MFI (right). Results represent individual mice (n = 8-9 mice per group) and mean ± SEM. (B) Flow cytometry analysis of p-AKT^S473 in plasma membrane–permeabilized cells from fractions A and B isolated from control (WT, red lines) and VAV-CRE (blue lines) mice, before (0) and 2 to 10 minutes after ex vivo addition of IL-7 (2 ng/mL) at 37°C. Quantitative expression of p-AKT^S473 normalized to WT with mean MFI at t = 0 (before IL-7 addition). Results are representative of 2 independent experiments for a total of 6 mice. (C) EBF1 protein expression was analyzed using flow cytometry after plasma and nuclear membrane permeabilization of control (WT, red areas and columns) and VAV-CRE (blue areas and columns) cells from fractions A and B. Representative MFI histograms of EBF1 protein expression (top). The quantitative expression of EBF1 protein after MFI normalization to WT with mean MFI (bottom). Results represent individual mice (n = 5-9 mice per group) and mean ± SEM. (D) Flow cytometry analysis of p-STAT5^Y694 in plasma membrane–permeabilized cells from fractions A and B isolated from control (WT, red lines) and VAV-CRE (blue lines) mice, before (0) and 2 to 10 minutes after the ex vivo addition of IL-7 (2 ng/mL) at 37°C. Quantitative expression of p-STAT5^Y694 normalized to WT with mean MFI at t = 0 (before IL-7 addition). Results are representative of a total of 6 mice. (E-F) Flow cytometry analysis of p-JAK1^Y1022 (E) and p-JAK3^Y980/981 (F) in plasma membrane–permeabilized cells from factions A and B isolated from control (WT, red lines) and VAV-CRE (blue lines) mice, before (0), 0.5, 1, 2, and 10 minutes after the ex vivo addition of IL-7 (2 ng/mL) at 37°C. Quantified expression of p-JAK1^Y1022 (E) and p-JAK3^Y980/981 (F) normalized to WT MFI at t = 0 (before IL-7 addition). Results are representative of 4 independent experiments for a total of 8 to 12 mice. P values were calculated using unpaired nonparametric t test. NS: P > .05; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. NS, nonsignificant; MFI, mean fluorescence intensity; SEM, standard error of the mean; WT, wild type.

This Feature Is Available To Subscribers Only