Notch1 signaling is detected in CLL cells. IgH.TEμ and age-matched wild type (WT) littermate control mice were analyzed at different disease stages to assess Notch1 signaling. (A) Representative flow cytometric analysis of total splenocytes stained for CD19, B220, and surface expression of Notch1 and Notch2 in WT littermate control and IgH.TEμ mice at indicated time points of 3 and 9 months (mos) are depicted. Histograms show Notch1 (upper) and Notch2 surface expression (lower) of splenocytes isolated from 3 (left) or 9-mos-old (right) IgH.TEμ (red line) and WT littermate control animals (blue line). Histogram profiles are gated on CD19⁺B220⁺ B cells for WT control and on CD19⁺B220⁻ CLL cells for IgH.TEμ animals. The dashed black line represents the fluorescence minus 1 (FMO) performed on the WT littermate controls omitting either Notch1 or Notch2 antibody staining. (B) Representative chemiluminescence profiles generated for quantification of GAPDH and NICD from whole cell extracts of sorted WT CD19⁺B220⁺ and IgH.TEμ CD19⁺B220⁻ splenocytes from 3-, 6-, and 9-mos-old animals using Simple Western System. (C) Quantification of relative protein expression of NICD comparing WT (n = 3) vs IgH.TEμ (n = 5) samples at 3 mos, WT (n = 3) vs IgH.TEμ (n = 3) samples at 6 mos, and WT (n = 3) vs IgH.TEμ (n = 3) samples at 9 mos using Compass Software (ProteinSimple). Quantification of flow cytometric analysis of relative values using mean fluorescence intensity (MFI) of intracellular Bcl2 expression in (D) spleen WT (n = 4) vs IgH.TEμ CD19⁺B220⁺ (n = 4) vs IgH.TEμ CD19⁺B220⁻ (n = 5) samples at 3 mos, WT (n = 3) vs IgH.TEμ CD19⁺B220⁺ (n = 4) vs IgH.TEμ CD19⁺B220⁻ (n = 3) samples at 6 mos and WT (n = 3) vs IgH.TEμ CD19⁺B220⁺ (n = 4) vs IgH.TEμ CD19⁺B220⁻ (n = 3) samples at 9 mos. (E) Quantification of LN from WT (n = 4) vs IgH.TEμ CD19⁺B220⁺ (n = 3) vs IgH.TEμ CD19⁺B220⁺ (n = 3) samples at 9 mos. P values were calculated using unpaired Student t test: *P < .05, **P < .01, and ***P < .001.