BCOR mRNA and protein expression in AML patients carrying BCOR disruptive mutations. (A) Box plots of BCOR mRNA levels quantified by real-time RT-PCR in 5 primary AML patients with BCOR disruptive mutations introducing premature stop codons (before the second last exon) compared with 14 BCOR-unmutated AML patients. All of the 5 BCOR-mutated AML patients display substantially decreased BCOR mRNA levels (P = .006 by Wilcoxon 2-sample test of ΔCt values). (B) Western blot analysis of BCOR protein expression in normal donor CD34⁺ hematopoietic cells (CD34⁺ I and CD34⁺ II, left panels) and in primary AML cells isolated from 15 patients with wild-type BCOR gene (right panels) showing a specific protein band of variable intensity corresponding to full-length BCOR (192 kDa predicted MW; arrow). (C) Western blot analysis of primary AML cells isolated from 5 patients harboring BCOR disruptive mutations (labeled in red: patient 326 with Thr733AlafsX5; patient 406II with Asn1485LysfsX5; patient 258A with Leu245ThrfsX19; patient 447A with Pro1115ThrfsX41; and patient 644A with His674MetfsX41). As a comparison, full-length BCOR expression in lysates from further 4 AML patients (patients 226, 157, 309, and 409) all devoid of BCOR mutations is shown. In the top panels, a specific protein band corresponding to full-length BCOR (192 kDa predicted MW) is observed (arrow) in wild-type BCOR AML, but not in BCOR-mutated AML. In patient 406II, a new faint band is detected (arrowhead) that likely corresponds to a truncated BCOR protein (162 kDa predicted MW). (B-C) The OCI/AML3 cell line not carrying BCOR mutations was used as a positive control for full-length BCOR protein expression. Protein lysate loading was evaluated by blotting the membranes with an anti-NPM1 or anti–β-tubulin Ab. Vertical lines have been inserted to indicate repositioned gel lanes.