HOXB4 HD contains the CUL4A-dependent degron. (A) A sequence alignment of the HDs of all HOX paralogous groups revealed a conserved LEXE motif. (B-C) Half-lives of HOXB4 LEXE mutants. Pulse-chase analysis of HOXB4 LEXE mutants (M1: L175D; M2: L175D, E176K; M3: L175D, E176K, E178K) was performed after transfection of the MYC-tagged proteins into HeLa cells, 35S-Met labeling, and immunoprecipitation with the anti-MYC antibody. The percentages of HOXB4 and the LEXE mutant proteins remaining at each time point were normalized relative to time 0 of each protein. The percentages of HOXB4 or LEXE mutant proteins remaining at each time point are indicated. (D) Determination of the steady-state levels of GFP-HOXB4 HD fusion protein in response to increasing MYC-CUL4A levels by immunoblotting with antibodies against GFP or MYC epitope tags, with β-actin as loading control.
