CUL4A targets HOXB4 for ubiquitin-dependent degradation. (A) Steady-state levels of MYC-HOXB4 were determined in response to increasing levels of cotransfected MYC-CUL4A in 293T cells by immunoblotting with the anti-MYC and β-actin (loading control) antibodies. (B) Pulse-chase analysis of HOXB4 half-life was performed following shCUL4A knockdown. (C) In vivo ubiquitination of HOXB4 in response to increasing levels of dominant-negative CUL4A was detected in 293T cells after tranfection with the indicated plasmids, treatment with MG132, precipitation with Ni²⁺-NTA agarose beads under denaturing conditions, and immunoblotting with anti-MYC antibody. (D) To determine the physiological effect of CUL4A and CUL4B on endogenous HOXB4 protein levels, bone marrow stem and progenitor cells from Cul4a and Cul4b knockout mice and their wild-type littermates were purified and analyzed by immunoblotting with anti-HOXB4. The upper panel shows the genotyping of Cul4a^−/− and Cul4b^−/Y mice by polymerase chain reaction.