Asrij interacts with and regulates CSN5 to maintain p53 levels in the BM. (A-D) Floxed and KO cells from 2-month-old mice were compared in all cases and are indicated in red and light green, respectively. (A) Immunostaining shows increased accumulation of polyubiquitinated proteins in KO BM cells compared with floxed controls (n = 30 cells from 3 mice per genotype). Maximum intensity projection of confocal microscopy images. (B) IP of endogenous p53 from whole BM shows increased levels of polyubiquitinated p53 in KO compared with asrij floxed. (C) RT-qPCR analysis for csn5 transcript levels in HSPCs (n = 3 mice per genotype). (D) Immunoblot analysis and quantification of CSN5 and MDM2 levels in HSPCs (n = 3 mice per genotype). (E) Surface and medial confocal planes of the same cell showing localization of Asrij (green) and CSN5 (red) in HSPCs (n = 30 cells from 3 mice per genotype). White dotted line marks the cell boundary. Plots show percent colocalization in surface and medial planes. (F) IP of endogenous Asrij in BM lysates from 2-month-old mice shows interaction with CSN5 but not with MDM2 or p53. (G-J) Lysates from HEK293 cells expressing FLAG-tagged Asrij constructs. (G) Asrij N- and C-terminal fragments were subjected to FLAG IP and probed for interaction with CSN5. At least 2 independent IP experiments were performed with similar results. (H-J) Asrij_FL-FLAG expressing cells (H) validated for FLAG expression by immunoblotting (n = 3), and for CSN5, MDM2, and p53 (I) transcript (J) and protein expression, as indicated. (A,E) Nuclei stained with DAPI (blue) and bars represent 2 µm. Statistically significant differences in transcript and protein levels determined using ANOVA: single factor analysis are indicated. Error bars denote standard error of mean. *P < .05, **P < .01 and ***P < .001. IP, immunoprecipitation.