Eμ-Tcl1 CLL spleen cells have elevated levels of Par-4 expression compared with normal B-cell subsets, which increases with progression of disease. (A) CLL cells were harvested from the spleen of Eμ-Tcl1 mice. B-cell subsets from C57BL/6J WT mice were isolated from the peritoneal cavity and spleen. Protein lysates of WT mice represent a pool of 20 mice. Levels of Par-4 and Tcl1 were quantified by western blot and normalized to β-actin. (B) Histogram shows relative Par-4 mRNA expression in Eμ-Tcl1 CLL cells and WT B-cell subsets quantified by qRT-PCR. Par-4 mRNA levels were normalized to mouse 18S mRNA expression and were then normalized to B-1a Par-4 mRNA expression. For panels A and B, results are representative of 2 experiments. Error bars represent standard error of the mean (SEM). *P < .05, **P < .001 as determined by the Student t test. (C) Par-4 protein expression increases with age and disease development in spleen cells isolated from the Eμ-Tcl1 mouse. Par-4 and Tcl1 protein expression is normalized to β-actin. Graphs show (D) correlation of Par-4 protein expression and percentage of CD5⁺CD19⁺ cells; (E) correlation of Par-4 protein expression and Tcl1 expression in the spleens of mice at specific ages. Each point represents an average of 3 mice. r² value determined by Pearson coefficient test and P = .0055 (D) and P = .8824 (E) determined by linear regression analysis. P. Cavity, peritoneal cavity.