Lack of GILZ enhances NF-κB transcriptional activity and Bcl-2 expression. (A) qPCR analysis of Bcl2, Bim, and Bmf mRNA expression in CD19⁺ BM cells cultured in vitro for the time indicated in the graph. (B) qPCR analysis of Bcl2 mRNA expression in CD19⁺ cells isolated from the BM of WT and KO mice (n = 6). Data are presented relative to the expression of Actb mRNA. (C) WB analysis of Bcl2 expression in purified B cells isolated from WT and gilz KO mice. (D) Immunohistochemical analyses show that the number of Bcl-2⁺ cells in spleen is increased in gilz KO compared with WT mice. Scale bars represent 100 µm; original magnification ×20. Insets in the upper right image represent higher magnification images (40×). (E) Images of in situ proximity ligation assay (PLA) performed on CD19⁺ cells isolated from the BM of WT mice. PLA was carried out in the absence (left) or presence (right) GILZ-p65/NF-κB primary antibodies mix. The right panel shows a representative merge of the red (PLA) and blue nuclear counterstain (DAPI) channels images. The red spots indicate close proximity (<40 nm) between bound antibodies. Scale bars represent 20 µm. (F) WB analysis of p65 expression in purified B cells isolated from WT and gilz KO mice. The same number of cells was loaded; WB with β-tubulin and laminin B antibodies served as loading control. The graph below represents densitometric analysis of NF-κB expression relative to housekeeping controls (n = 2). (G) Chromatin IP assay of NF-κB binding on proximal promoter of Bcl2 in WT and gilz KO CD19⁺ cells isolated from BM. Cell lysates were immunoprecipitated with anti–NF-κB or control IgG, and the presence of specific regions in the immunoprecipitates was determined by qPCR. Graphs represent mean ± SEM. *P < .05, **P < .005, ***P < .0005.