TRIM33 regulates A-MYB stability through ubiquitination.

<p>(<b>A</b>) HEK293T cells were transfected with HA-tagged ubiquitin and FLAG-tagged A-MYB in the presence or in the absence of GFP-tagged TRIM33. To detect ubiquitinated A-MYB, immunoprecipitation was performed on the lysates using anti-FLAG affinity beads and a Western blot was performed using anti-HA antibodies (top panel). To detect the immunoprecipitated A-MYB, the Western blot was probed with anti-FLAG antibodies (middle panel). Expression of tagged TRIM33 and the loading control HSP70 is shown in the bottom panel. (<b>B</b>) HEK293T cells were transfected with HA-tagged ubiquitin and FLAG-tagged A-MYB and either a control or TRIM33 shRNA expression vector. A-MYB was immunoprecipitated and Western blotting was performed as in (<b>A</b>) to detect ubiquitinated A-MYB (top panel). Lysates were subjected to Western blotting with anti-TRIM33 antibody to show knock down and anti-γ-Tubulin antibody as a loading control (bottom panels). (<b>C</b>) The protein stability of A-MYB in the presence and in the absence of TRIM33 was monitored using the protein translation inhibitor cycloheximide (CHX). Cells were transfected as in (<b>A</b>) and CHX was added at 10 μg/ml and samples were analyzed by Western blots at various time points, as shown. HSP70 was used as the loading control. (<b>D</b>) FLAG-tagged A-MYB and GFP-tagged TRIM33 were co-expressed in HEK293T cells. Lysates were subjected to immunoprecipitation with anti-FLAG beads or, as a control, anti-IgG beads, and co-immunoprecipitation of TRIM33 with A-MYB was detected by Western blotting with anti-GFP and anti-FLAG antibodies, respectively (top panels). Expression of tagged Trim33 in lysate is shown by Western blotting with anti-GFP antibody and as a loading control anti-HSP70 is also shown (bottom panels).