Fig. 2.

Chk1 is essential for early B cell development. a Relative Chk1 mRNA levels during B cell development were assessed by analyzing RNA-sequencing data (left) and validated by real-time qPCR (right). RNA-Seq analysis was performed using pooled RNA samples derived from FACS-sorted bone marrow pro-B (B220⁺ μHC⁻ ckit⁺), large pre-B (B220⁺ μHC⁻ CD25⁺ FSC^high), small pre-B (B220⁺ μHC⁻ CD25⁺ FSC^low) and immature B cells (B220⁺ μHC⁺ κHC⁻) as well as splenic mature B cells (B220⁺) left untreated or treated with 1 μg/ml anti-CD40 antibody for 48 h that were isolated from wild-type C57Bl/6N mice (n = 4). Bars depict the relative number of sequence reads, normalized to the pro-B cell stage (arbitrary set to 100%). Real-time qPCR: analysis was performed using RNA samples derived from FACS-sorted bone marrow pro B (B220⁺ CD19⁺ AA4.1⁺ μHC⁻ CD25⁻ ckit⁺), large pre B (B220⁺ CD19⁺ AA4.1⁺ μHC⁻ CD25⁺ ckit⁻ FSC^high), small pre B (B220⁺ CD19⁺ AA4.1⁺ μHC⁻ CD25⁺ ckit⁻ FSC^low) and immature B cells (B220⁺ CD19⁺ AA4.1⁺ μHC⁺) as well as splenic mature B cells (B220⁺ CD19⁺) left untreated or treated with 1 μg/ml anti-CD40 antibody for 48 h that were isolated from wild-type C57Bl/6N mice (n = 3). b Western analysis showing CHK1 protein expression throughout B cell development. Samples from three C57Bl/6N mice were pooled for analysis. BM pre + pro B cells: B220⁺ CD19⁺ AA4.1⁺ IgM⁻, BM immature B cells: B220⁺ CD19⁺ AA4.1⁻ IgM⁺, mature B cells (B220⁺ CD19⁺) from spleen were lysed immediately or treated with 1 μg/ml anti-CD40 antibody for 48 h. c Spleen weight and representative pictures (scale bar = 5 mm) of spleens of 6–8 weeks old mice of the indicated genotypes. d Quantification of flow cytometric analyses defining TCRβ⁺ T and B220⁺ B cell populations in spleens of the indicated genotypes. e Flow cytometric analysis of bone marrow resident B cells in mice of the indicated genotypes. Bars in c–e represent means of n = 5 WT, n = 3 Mb1-Cre ⁺, n = 5 Mb1-Cre ⁺ Chk1 ^fl/− ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t test