Cul4A-DDB1 E3 ligase promotes CRY1 ubiquitination both <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A</b>) Enhanced CRY1 ubiquitination in the presence of CUL4A-DDB1 E3 ligase. 293T cells were co-transfected with Cry1-Flag and Myc-ubiquitin in the presence of GFP, HA-Cul4A plus T7-Ddb1, or HA-Cul4A-DN plus T7-Ddb1. 24 hr after transfection, cells were treated with proteasome inhibitor MG132 (5 μM) for 16 hr and then harvested for detection of CRY1 ubiquitination. The protein levels of CRY1, CUL4A, DDB1 and DN-CUL4A in the whole cell lysates were determined by immunoblotting with specific antibodies. All the experiments were repeated at least three times and a representative result was shown here. (<b>B</b>) <i>In vitro</i> CRY1 ubiquitination by CUL4A-DDB1 E3 ligase. The purified GST-CRY1 fusion protein was mixed with the DDB1-CUL4A complex isolated from adenovirus-transduced 293T cells in the presence of E2, E3, and ubiquitin at 37°C for 2 hr before immunoblotting with anti-ubiquitin. GST-CRY1 and GST control were shown by Coomassie blue staining.(<b>C</b>) Reduced CRY1 ubiquitination in <i>Ddb1-LKO</i> primary mouse hepatocytes. After isolation from either 8-wk old <i>Ddb1</i>^{<i>flox/flox</i>} or <i>Ddb1-LKO</i> mice, primary mouse hepatocytes were transduced with Ad-Cry1-Flag for 24 hr and treated with MG132 (5 μM) for 16 hr before harvest. The protein lysates were subjected to denaturing IP to detect CRY1 ubiquitination. The protein levels of CRY1 and DDB1 in the whole cell lysates were measured by immunoblotting with either anti-FLAG or anti-DDB1. All the experiments were repeated three times and the representative result was shown here. (<b>D</b>) Adenoviral overexpression of <i>Ddb1</i> rescues CRY1 ubiquitination in <i>Ddb1-LKO</i> primary mouse hepatocytes. Primary mouse hepatocytes were isolated from <i>Ddb1-LKO</i> mice and transduced by Ad-Cry1 along with either Ad-GFP or Ad-Ddb1. 24 hr post-transduction, cells were treated with MG132 for 8 hr and then harvested for detecting CRY1-ubiquitination.