Par-4 downregulates p65-mediated NF-κB signaling. (A) Whole splenocytes from 3- to 4-month-old WT mice mounted, fixed, and stained with anti-B220-APC-Cy7, DAPI, rabbit anti-p6,5 and anti-rabbit-AF488 secondary after stimulation with LPS. (B) Representative images of single, B220⁺ cells measured for colocalization of AF488-labeled p65 with DAPI stain with and without LPS stimulation. (C) Colocalization of AF488 and DAPI stains, digitally quantified by Imaris imaging software using the Manders coefficient, was shown to increase in a dose-dependent manner after LPS stimulation. (D) Whole splenocytes from 3- to 4-month-old TCL1 and Par-4 × TCL1 mice were mounted, fixed, and stained with anti-B220-APC-Cy7, DAPI, rabbit anti-p65 and anti-rabbit-AF488 secondary. Cells selected by B220 positivity were measured for colocalization of p65 signal with DAPI stain to determine nuclear localization of p65. (E) Representative images of single B220⁺ cells from both genotypes measured for colocalization of AF488-labeled p65 with DAPI stain. (F) Representative colocalization images of single 60× fields containing 60 cells from TCL1 and Par-4 × TCL1 mice showing less p65 colocalization with DAPI stain in Par-4 × TCL1 mice. (G) Quantification of colocalization by Imaris imaging software through use of the Manders coefficient (P = .0267, n = 5). Results were tested for significance in an unpaired Student t test with α set to 0.05. Original magnification ×60 for panels A-B,D-F.