Increase in IRF4 levels in PB-committed cells is concomitant with a downregulation of CBLB expression. (A) CBL, CBLB, and IRF4 mRNA expression levels as measured by RNA-seq in the different cell populations (n = 3 paired samples). (B) IRF4, CBLB, and CBL protein expression assessed using western blots in NBCs, D4 cells, and different cell subsets on D6. The western blot (WB) is representative of 2 independent experiments. (C) IRF4 protein expression after treatment with cycloheximide (CHX; 50 µg/mL) to inhibit protein synthesis at D6. We could not detect any degradation of IRF4 after 4 hours. Positive control: p21, a protein known to be degraded by the proteasome pathway. Data are representative of 3 experiments. (D) IRF4 protein expression after MG132 treatment (10 µM for 4 hours) to inhibit proteasome degradation at D6. We could not detect any accumulation of IRF4 protein. Positive control: p21. Data are representative of 3 experiments. (E) Ubiquitination of IRF4 after ubiquitin immunoprecipitation in activated B cells (D4lo plus 8 hours of IL-2, IL-4, and IL-10). Western blots of IRF4 (left), with an enrichment in IRF4 in immunoprecipitated ubiquitin lysate compared with input and control and of ubiquitin (Ub; right) as control. Data are representative of 2 experiments. (F) CBLB detection by western blot after IRF4 immunoprecipitation in activated (D4 plus 14 hours) B cells. Although CBLB and IRF4 could be detected in input lysate and immunoprecipitation of IRF4 was successful (enrichment compared with input), CBLB could not be detected as interacting with IRF4. Data are representative of 2 experiments. (G) An IGV browser window showing RNA-seq and ATAC-seq signals close to the start of the CBLB locus in the different cell populations. Asterisks denote the first 3 exons of CBLB. The red box indicates an ATAC-seq region whose density is correlated with CBLB expression and that is known to interact with the CBLB promoter (according to FANTOM5³⁶ ). The orange box indicates the promoter and the first intronic regions, including the ATAC-seq regions. They acquired the repressive H3K27me3 mark at the pre-PB stage of differentiation, and they kept it at the PB stage⁵¹  (purple and green rectangles, respectively). Ig CTL, immunoglobulin control.