FBXO11 binds, ubiquitinates, and destabilizes BAHD1. (A) Quantitative transcriptome (top graph, 14 642 mRNAs) and proteome (bottom graph, 9822 proteins) comparisons of WT and FBXO11 KO (F11KO, panel G) HUDEP-2 cell pools. Each dot represents a single gene (average values: WT, 4 biological replicates; F11KO, 3 biological replicates each for FBXO11, sgRNA1, and sgRNA2). The x-axis shows the log₂ fold change (FC) in expression. The y-axis shows the –log₁₀P value. Selected erythroid genes are highlighted. (B) Gene set enrichment analysis (GSEA) of mRNAs that are downregulated in F11KO vs WT cells, showing enrichment for erythroid differentiation markers²⁴  and erythroid GATA1 targets.²⁵  (C) GSEA of mRNAs that are upregulated in F11KO vs WT cells, showing enrichment for T-cell and hematopoietic stem/progenitor cell markers. (D) Integrative analysis of quantitative proteomics (9822 proteins) and RNA-seq (14 642 mRNAs) comparisons of WT and F11KO HUDEP-2 cell pools grown in culture under expansion conditions. Each dot represents the average value for 1 gene or protein (from 4 biological replicates for WT; 6 for F11KO cells). The erythroid transcription factors GATA1, KLF1, and TAL1 are indicated in blue. Potential FBXO11 substrates were identified as those with a ≥1.6-fold increase in protein expression (log₂ FC ≥0.7; P < .05) and a minimal change in the corresponding mRNA (log₂ FC ≤0.5) (see supplemental Methods). The size of the black dot represents the relative P value in quantitative proteome analysis (see supplemental Table 4). (E) Western blot showing the expression of selected proteins identified in panel D. (F) Western blot showing BAHD1 expression in primary erythroblasts derived from CD34⁺ cells electroporated with ribonucleoprotein containing Cas9 and control (Ctrl) or FBXO11 sgRNAs. (G) WT and F11KO HUDEP-2 cells (clone F11KO #2) stably expressing BAHD1-MYC were treated with MG132 or vehicle, immunoprecipitated (IP) with anti-MYC antibody, and analyzed by western blotting. The input lanes represent 10% of the immunoprecipitated sample. (H) Primary structure of full-length (FL) BAHD1, showing the central proline-rich region (cPRR), nuclear localization signal (NLS), and bromo-adjacent homology domain (BAH). N- and C-terminal truncated mutants are shown below. Plasmids encoding MYC-tagged versions of BAHD1 or GFP were transfected into 293T cells. After 48 hours, cell lysates were immunoprecipitated with anti-MYC antibody, and western blot analysis was performed with anti-FBXO11 or anti-MYC antibodies. The input lanes represent 10% of the immunoprecipitated sample. (I) The ubiquitin-activating enzyme UBA1 was incubated with fluorescent-tagged ubiquitin and E2 ligase UBCH5B to form the intermediate UBCH5B∼UB (ubiquitin). The indicated recombinant BAHD1 N-terminal fragments were preincubated with NEDD8∼CUL1 ± SKP1-FBXO11, followed by the addition of UBCH5B∼UB. At the indicated time points, aliquots were quenched by adding sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and fractionated on a 4% to 12% SDS-polyacrylamide gradient gel. Fluorescent UB was visualized by scanning on a Typhoon imager (GE). FDR, false discovery rate; NES, normalized enrichment score.