Figure 5. Elevated activation of the NOTCH pathway in IRF4-deficient B cells. (A) irf4+/+ and irf4−/− splenic B cells were analyzed for expression of HES5 and DTX1 by qRT-PCR. The expression was corrected for β-actin levels and then normalized to wild-type B cells. Data are shown as mean ± SD (n = 3). Statistical significance was determined by Student’s t test (*, P < 0.05; ***, P < 0.001). (B) NOTCH2 expression in irf4+/+ and irf4−/− splenic B cells was analyzed by Western blot (whole cell lysates; one representative of three independent experiments). (C) NOTCH2 expression on MZ and FO B cells from irf4+/+ and irf4−/− mice was analyzed by flow cytometry (gates are indicated on the corresponding dot plots). Data are shown as average mean fluorescence intensity (MFI) value ± SD (n = 3; top, irf4−/−; bottom, irf4+/+). Statistical significance was determined by Student’s t test (*, P < 0.05; **, P < 0.01). (D) NOTCH2 mRNA expression in irf4+/+ and irf4−/− splenic B cells was analyzed by qRT-PCR as described in A. Data are shown as mean ± SD (n = 3). (E) NOTCH2 protein expression in the cytosolic/membranous (C + M) and nuclear (N) protein fractions of irf4+/+ and irf4−/− B cells was determined by Western blot (one representative of two independent experiments). Lamin B1 and α-tubulin were used as loading controls for N and C + M fractions, respectively. (F) NOTCH2, MOMA1, and B220 expression in spleen sections from irf4+/+ and irf4−/− mice was analyzed by IF. Arrows indicate B220+ B cells expressing nuclear NOTCH2. One representative mouse out of four per group is shown. (right) Specificity of the NOTCH2 staining was confirmed by isotype control staining on consecutive sections. (G) NOTCH2 and B220 expression in the spleen of irf4fl/flCD20-TAM-Cre and irf4fl/+CD20-TAM-Cre mice was analyzed by IF. Nuclei were stained with DAPI. Dashed lines mark the inner and outer border of the MZ area. Arrows indicate B220+ B cells expressing nuclear NOTCH2. One representative mouse per group is shown (irf4fl/flCD20-TAM-Cre: n = 6; irf4fl/+CD20-TAM-Cre: n = 8; four independent experiments). (F and G) Bars, 50 µm. (H) NOTCH2 expression on eGFP+ MZ B cells of irf4fl/flCD20-TAM-Cre and irf4fl/+CD20-TAM-Cre mice was analyzed by flow cytometry. Values on plot indicate average MFI value ± SD (n = 3–4, two independent experiments; top, irf4fl/fl; bottom, irf4fl/+). The statistical significance was determined to be P = 0.06 by Student’s t test. (I) irf4+/+ and irf4−/− splenic B cells were stimulated with α-CD40 alone or in combination with α-IgM for 48 h, and NOTCH1 activation was assessed by Western blot. Control cells (CNTRL) were cultured for 4 h in medium only. The figure shows one representative of three independent experiments. (J) FBXW7 expression in irf4+/+ and irf4−/− splenic B cells was assessed by GEP. Data are shown as mean ± SD (n = 4). Statistical significance was determined by Student’s t test (**, P < 0.01).
Figure 5.

Elevated activation of the NOTCH pathway in IRF4-deficient B cells. (A) irf4+/+ and irf4−/− splenic B cells were analyzed for expression of HES5 and DTX1 by qRT-PCR. The expression was corrected for β-actin levels and then normalized to wild-type B cells. Data are shown as mean ± SD (n = 3). Statistical significance was determined by Student’s t test (*, P < 0.05; ***, P < 0.001). (B) NOTCH2 expression in irf4+/+ and irf4−/− splenic B cells was analyzed by Western blot (whole cell lysates; one representative of three independent experiments). (C) NOTCH2 expression on MZ and FO B cells from irf4+/+ and irf4−/− mice was analyzed by flow cytometry (gates are indicated on the corresponding dot plots). Data are shown as average mean fluorescence intensity (MFI) value ± SD (n = 3; top, irf4−/−; bottom, irf4+/+). Statistical significance was determined by Student’s t test (*, P < 0.05; **, P < 0.01). (D) NOTCH2 mRNA expression in irf4+/+ and irf4−/− splenic B cells was analyzed by qRT-PCR as described in A. Data are shown as mean ± SD (n = 3). (E) NOTCH2 protein expression in the cytosolic/membranous (C + M) and nuclear (N) protein fractions of irf4+/+ and irf4−/− B cells was determined by Western blot (one representative of two independent experiments). Lamin B1 and α-tubulin were used as loading controls for N and C + M fractions, respectively. (F) NOTCH2, MOMA1, and B220 expression in spleen sections from irf4+/+ and irf4−/− mice was analyzed by IF. Arrows indicate B220+ B cells expressing nuclear NOTCH2. One representative mouse out of four per group is shown. (right) Specificity of the NOTCH2 staining was confirmed by isotype control staining on consecutive sections. (G) NOTCH2 and B220 expression in the spleen of irf4fl/flCD20-TAM-Cre and irf4fl/+CD20-TAM-Cre mice was analyzed by IF. Nuclei were stained with DAPI. Dashed lines mark the inner and outer border of the MZ area. Arrows indicate B220+ B cells expressing nuclear NOTCH2. One representative mouse per group is shown (irf4fl/flCD20-TAM-Cre: n = 6; irf4fl/+CD20-TAM-Cre: n = 8; four independent experiments). (F and G) Bars, 50 µm. (H) NOTCH2 expression on eGFP+ MZ B cells of irf4fl/flCD20-TAM-Cre and irf4fl/+CD20-TAM-Cre mice was analyzed by flow cytometry. Values on plot indicate average MFI value ± SD (n = 3–4, two independent experiments; top, irf4fl/fl; bottom, irf4fl/+). The statistical significance was determined to be P = 0.06 by Student’s t test. (I) irf4+/+ and irf4−/− splenic B cells were stimulated with α-CD40 alone or in combination with α-IgM for 48 h, and NOTCH1 activation was assessed by Western blot. Control cells (CNTRL) were cultured for 4 h in medium only. The figure shows one representative of three independent experiments. (J) FBXW7 expression in irf4+/+ and irf4−/− splenic B cells was assessed by GEP. Data are shown as mean ± SD (n = 4). Statistical significance was determined by Student’s t test (**, P < 0.01).

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